Journal of Applied Biology & Biotechnology Vol. 10(03), pp.

163-176, May, 2022

Available online at http://www.jabonline.in
DOI: 10.7324/JABB.2022.100321

Techniques in scaffold fabrication process for tissue engineering

applications: A review

Abinash Kumar, Anu Jacob*

Department of Biotechnology, Karunya Institute of Technology and Sciences, Coimbatore, India.

ARTICLE INFO ABSTRACT

Article history: Tissue engineering is a highly complex process with goals to replace, restore, and regenerate tissues. Tissue
Received on: August 03, 2021 engineering combines multidisciplinary fields such as biochemistry, clinical medicine, biological science, and
Accepted on: December 15, 2021 materials science. It has application in personalized drugs, organ transplantation, and as a drug transporter.
Available online: April 10, 2022 The scaffold fabrication process for tissue engineering depends on numerous factors such as biodegradability,
mechanical possessions, scaffold architecture, and manufacturing process. The scaffold properties based on
its biological aspects, structural requirements, material composition, conventional and advanced fabrication
Key words:
technologies, and extrusion-based scaffold fabrication techniques are analyzed and discussed in the current
Biocompatibility, biodegradability,
review. Further studies for the development of bio-scaffolds will provide a broader roadway into a new
conventional methods, scaffold,
dimension of various tissue engineering techniques and provide greater advancement in medical and clinical
tissue engineering
research.

1. INTRODUCTION various pathogens with minimum inflammation, and toxicity

Tissue engineering is an interdisciplinary field that connects rate. In the 3D scaffolds fabrication technique, the materials are
biomedical engineering, mechanical engineering, clinical generally classified into chemical factors such as synthetic and
medicine, genetic engineering, and biotechnology. It is a complex natural polymers, hydrogels, metals and non-metals, composites,
process as tissue regeneration depends upon various factors such as ceramics, and non-ceramics [7]. The development of biologically
maintenance of cell-cell interactions, surface properties, porosity, synthesized scaffolds depends on various factors such as pore
mechanical stability, solubility, and degradability of biomaterials sizes, interconnectivity between the pores, biodegradability, ability
used. Accidents and conditions, such as osteomyelitis, arthritis, for cells to produce their extracellular matrix, machine limitation,
anaemia, cancers, hereditary multiple exostoses, and hereditary biocompatibility, clinical status, instrumentation choice, good
bone marrow failure syndromes, cause severe damage to the bones manufacture practices, and mechanical properties. In addition,
cartilage and tissues[1–5]. With the increase in demand for bone the modifications considered during fabrication of bio-scaffolds
grafts in organ transplantation and substitute surgery, the market are bio-mimicking with various cellular components, delivering
value is expected to be 11.5 billion US dollars by the end of 2025 of different bioactive molecules and ameliorative agents like
[6]. Tissue engineering is the development of porous scaffolds to antibiotics, cytokines, drugs, inhibitor, growth factors, proteins,
provide a favourable environment for the regeneration, growth which provide an anchorage for great importance in stacking to the
of tissues, and complex organs. Scaffolds are three-dimensional scaffolds [8,9]. With the advancement in technology and various
structures that are porous, fibrous and permeable biomaterials. architectural demands, various approaches have been developed
It helps in the transport of body fluids, promotes cell-cell for the fabrication of scaffold materials.
interaction, deposition of extracellular matrix, viability against Although the conventional methods are widely used and are
evolving with decades, advanced prototyping techniques are also
being adopted. The advanced techniques use high- end computer-
*Corresponding Author aided designing software for the bio fabrication process with
Anu Jacob, Department of Biotechnology, Karunya Institute of Technology micro, macro, and nanoscale architecture [7,8]. With the recent
and Sciences, Coimbatore, India. E-mail: anujacob @ karunya.edu development of biologically active agents, natural biopolymers
© 2022 Kumar and Jacob. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlike
Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
164 Kumar and Jacob: Journal of Applied Biology & Biotechnology 2022;10(03):163-176

Table 1: Type of materials used in scaffolds fabrication.

Types of bio-
Examples Advantages Disadvantages Application Fabrication technique
materials
Inkjet printing, gel casting
Hydroxyapatite, β-TCP, Slow degradation
Non-toxic, biocompatible, binder, salt leaching, SLS,
α-TCP, calcium silicate, rate, low mechanical Bone and dental
Ceramics anti-inflammatory, osteo- stereolithography, extrusion
calcium sulphate, TCP, strength, compact in tissues applications
conductive. type, fused deposition
bioactive glasses. nature, brittle.
modelling.
Poor osteo-integration
Tantalum, Co–Cr, Iron
with the nearby bone. Bone, and dental
magnesium alloys, Non-toxic, corrosion SLS, stereo lithography,
non-biodegradable, application, knee
Metals and Mg-RE Mg-Ca, Fe-Mn resistance, light-weight, vacuum foaming, electron
release toxic ions replacement surgeries
alloys alloys, stainless steel, biocompatible, osteo- beam melting, selective laser
which causes and artificial hearing
Fe foam, Titanium, Ti, conductive melting.
inflammatory applications
Ti–6Al–4V.
responses
CS, alginate, dextran, Biodegradable, non-toxic, Rapid degradation,
collagen, gelatine, biocompatible, support complex structure, Drug delivery, Electro-spinning,
glycosaminoglycan, cell-cell attachment and less mechanical bone and tissue inkjet printing, solvent
Natural polymers
agarose fibrinogen, differentiation, anti- strength, water- applications, gene casting, emulsification,
actin, keratin, cellulose, allergenic, osteo-conductive, soluble, less cell therapy photolithography.
hyaluronic acid. viscoelastic. attachment
Polyesters, Biodegradable, good
Freeze drying, electro
polycarbonates mechanical strength, Bone, tissues,
Toxic, hydrophobicity, spinning, gas foaming,
polyether, polyhydroxy biocompatibile, non-toxic, cartilage and dental
high production cost, electron beam melting,
Synthetic acids, polysiloxanes, low melting point, easy to application, drug
slow degradation, selective laser melting phase
polymers polylactones, PCL, manufacture and design, delivery
antigenic, less cell- separation, fused deposition
polyurethanes, the unwanted product can
cell interaction. modelling, SLS, stereo
polyorthoesters be degraded and can be
lithography.
polyanhydride. removed
Different polymers,
ceramics and metals
are blended. Calcium
silicate, calcium Good mechanical strength,
Slow degradation, less Bone, tissues, Freeze-drying method, fused
sulphate, tricalcium, biocompatibile, non-toxic,
Composites cell-cell interaction, cartilage and dental deposition modelling, stereo
iron magnesium osteo- conductive, corrosion
compact in nature. application lithography.
alloys, CS, alginate, resistant, lightweight
dextran, collagen,
gelatine, polysiloxanes,
polylactones.

are replacing synthetic polymers. The natural polymers are biomaterials used in regeneration and restoration applications,
either plant-derived or animal-derived and are biodegradable, have some disadvantages such as scarcity, expensiveness, high
non-toxic, and provide customized pore sizes and renewability cell deaths, and poor biocompatibility [11].
[10]. Moreover, the various types of natural bio-polymers play
This review describes different techniques of advanced rapid
an important role in biocompatibility, influence cell behaviour,
prototyping (RP) and conventional 3D fabrication for bio-scaffolds
possess high surface area, with pre-existing vascular networks,
preparation. These techniques can create porous 3D structures
porosity, and rapid biodegrading properties [11].
with controlled mechanical possession, pore size, interconnected
The biomaterials and fabrication methods are selected for pores, and porosity. The paper summarizes research status of each
processes depending upon the analysis and complexity of targeted of the methods and the opportunities and challenges are analyzed.
tissues. Different type of materials used in scaffolds fabrication for
various tissue engineering applications is compiled in the Table 1. 2. FABRICATION OF SCAFFOLDS BASED ON THEIR
Recently, various studies reported on advancement in biomaterials REQUIREMENTS
fabrication technique and development of scaffolds from The scaffold fabrications and designing consider various properties,
biomaterials which enhanced cell proliferation, cell viability, such as mechanical, biological, and physicochemical based on its
and printability without any stress [7,12–15]. Plant-based feasibility and requirements. Additionally, the interconnectivity
biomaterial such as plant proteins, lignin, polysaccharides, within the pores, shape, pore size, porosity, strength, and
and plant extracts have various bioactive products that are degradation rate are the important factors based on which the
useful in various restoration, regeneration and improvement of scaffolds fabrications depend. The 2D scaffolds technique
scaffolds fabrication. However, the synthetic and animal-derived possesses several advantages by providing higher resolution and
 Kumar and Jacob: Techniques in scaffold fabrication process for tissue engineering applications 2022;10(03):163-176 165

accuracies with control over physical and chemical properties. The design of scaffold for tissue engineering involves high
The imaging and characterization are easier with automated lab interconnectivity within the nano-vascular networks of
facilities and high processing screening methodologies [16,17]. extracellular matrix, transport of oxygen, nutrient, and various
The 3D bio fabrication techniques for scaffolds design uses soluble factors that are responsible for removing metabolic wastes
advanced bio-printing and bio-assembly methodology that include [7,11]. Based on its complexity, construct materials source,
cells for the fabrication process in an automated manner [18]. geometrical distribution of structure and the process of fabrication
It uses various types of computer-assisted designing software technique, the 3D scaffolds are available in various forms Table 3.
packages to create virtual cross-sections of cell-loaded matrices
by consecutive layers formation with a computational fabrication 3. DIFFERENT TYPES OD SCAFFOLD FABRICATION
process. The 3D scaffold designs are a form of sponges, foams, TECHNIQUES
and meshes and can resist the external pressure caused by various
The conventional tissue engineering scaffold production
factors such as different tissue interaction with the extracellular
techniques include thermally induced phase separation (TIPS),
matrix, mechanical stiffness, rapid degradation, cell deaths,
fiber bonding, electrospinning, solvent casting and particulate
toxicity, and biocompatibility. Nevertheless, the designing of 3D
leaching, membrane lamination, freeze-drying, and gas foaming
scaffolds develops proper homogeneous mixture, cell-to-cell
[49]. The recent development in scaffold fabrication mainly
contacts, cell proliferation and cell attachment. The emerging 3D
comprises integration with computer-aided design (CAD)
technology has revolutionized into 4D printing depending on the
software through RP technology such as stereolithography, bio-
type of biomaterials and environmental factors [19]. However, the
plotting, solvent-based free forming, combination modelling
4D printing technique is quite expensive as compared to other 3D
technique, fused deposition modelling, 3D printing, and selective
printing technologies [20]. The scaffolds prints are prepared with
laser sintering (SLS) [8,9]. The techniques retain the ability
advanced processing through multiscale finite element analysis and
to maintain pore structures, cell–cell interaction, reduction in
computational neuromusculoskeletal evaluation to obtain load-
mechanical instability, and control over mitigation of the cellular
holding capacity, in vivo cyclic stress and biocompatibility [21].
matrix. Advantages and disadvantages of different type scaffolds
Different types of scaffolds and fabrication techniques are used fabrication technique for tissue engineering applications are
depending upon their biomaterials’ composition (Table 2). For compiled in Table 4.
example, in bone tissue engineering, nanofibrous scaffolds are
used that mimics extracellular matrices and collagen fibres [32– 4. CONVENTIONAL METHODS
34]. Gelatin and Fibrin are natural biopolymers that have been There are various conventional methods of scaffold fabrication (Fig.
widely used in scaffold fabrication as it has high biodegradability 1) and have been developed for drug delivery, 3D cell culture and
and biocompatibility [35,36]. Alginate has been widely used for tissue engineering. However, the conventional scaffold fabrication
bone and cartilage tissue engineering and capable of scaffold- techniques sometimes remain incompatible as they deviate from
reinforcement and non-immunogenic property [37]. the optimal environment for cell attachment, multiplication, and

Table 2: Different scaffolds fabrication for various tissue engineering applications.

Biomaterial composition Technique Cells type used Result References
Poly (ethylene glycol)
Inkjet printing Articular chondrocytes Cartilage tissue formation [22]
dimethacrylate hydrogels
Tissue engineering
PCL/gelatin scaffold Electrospinning L929 mouse fibroblast cells [23]
applications
Hydrogel-filled polylactide Thermally-induced phase Bone and cartilage tissue
Chondrocytes [24]
porous scaffold separation engineering
3D bioprinting nanocellulose
Extrusion Chondrocytes Cartilage tissue engineering [25]
and alginate scaffolds
Poly (ethylene glycol) Vascular endothelial growth
Stereo-lithography NIH-3T3 [26]
dimethacrylate hydrogels factor secretion
Alginate-PVA-hydroxyapatite Best for osteo-conductivity
Bioprinting MC3T3 [27]
hydrogel and tissue engineering
Laminated hydroxyapatite
Cellular regeneration and
nanoparticle layer on
Electrospinning MSCs attachment, osteogenic [28]
polyhydroxybutyrae fibrous
phenotypic generation
scaffold
CS polyelectrolyte complex Freeze drying Good cytocompatibility and
feline fibroblast cells [29]
porous scaffolds Cartilage tissue engineering
Hydrothermal cross-linked
Freeze drying L929 mouse fibroblast cell Cartilage tissue engineering [30]
CS porous scaffolds
Electrospun nanofibrous
Swiss mouse NIH 3T3
polyurethane/poly(glycerol Electrospinning Vocal fold tissue engineering [31]
fibroblasts
sebacate) scaffolds
166 Kumar and Jacob: Journal of Applied Biology & Biotechnology 2022;10(03):163-176

Table 3: Various forms of 3D scaffolds.

Types of scaffolds Materials Advantages Disadvantages References
High porosities and homogeneous
Poly(lactic-co-glycolic acid),
interconnected pore structure, nutrient [38,7]
poly-L-lactic acid, PCL,
Porous scaffolds and gas transportation through Pore size dependant
polybutylene terephthalate,
channel networks, yield space for the
polyglycolic acid
extracellular matrix with the cells
Great potential for neurite growth by
depending on cell separation, mimic
Poly(lactic) acid, Poly- Limited material
the extracellular matrix depending on
Fibrous scaffolds caprolactone, cellulose, silk selection, inadequate [39,40]
surface volume ratio, porosity and cell
fibroin, gelatine, collagen resolution
infiltration, induces greater cellular
attachment as compare to microfibers
Have controlled architecture and
Limited to photoresist,
reproducible properties, high precision
Solid free-form Synthetic and natural residual toxic moieties,
geometrically complex scaffolds with [7,41]
scaffolds biopolymers post-processing
controlled pore size forming high
challenges
interconnectivity within the pores
Inexpensiveness, high surface areas,
chemical signalling, biocompatibility,
porosity, degradable, vascular Low immunogenicity
Soya protein, camelina protein, networks, liable mechanical potentials, low
Natural-biopolymer
gluten, zein cellulose, pectin, characteristics, high polarity and molecular weight, time- [7,11]
scaffolds
starch, lignin, plant extracts due to hydrophilic potentialities, consuming. less dense,
greater cell attachment effects, greater chemical complexity
mimics capacity with the extracellular
matrixes.
Limits to form a
proper extracellular
Has great control over the architecture
Alloplastic-synthetic Synthetic polymers, glass, matrix, degradation of
of the construct and various [7,42]
scaffolds ceramics, metals scaffolds, toxicity, poor
mechanical properties
biocompatibility and
expensive in natures
Gelatine, fibrin, agarose, Semi-crystalline, amorphous,
Have limited
alginate, synthetic polymers, high flexibility, biocompatibility,
Hydrogel scaffolds mechanical properties, [7,43]
natural polymers, Fibrinogen, hydrophilicity, degradability, abled to
small pore size
collagen, CS, hyaluronic acid survive in harsh environment
Cells tendency depends
Widely used for gene therapy, tissue
Micro-sphere Collagen, CS, gelatine, poly- upon the type of
engineering, site-specific drug [44,45]
scaffolds lactic acid-glycolic acid materials and methods
delivery, growth factors
incorporated, costly
Good compression and corrosion Reliability, slow
Bio-ceramics scaffold α-TCP, β-TCP, bioactive glass resistance, bio-resorbability and good degradation rate, poor [46,47]
biocompatibility fidelity
Great biocompatibility and Poor cell affinity and
Polymer-ceramic PLA, polylactic co-glycolic
biodegradation, absorbability, high cell-matrix interaction, [46,48]
composite scaffold acid, Polyglycolic acid
toughness, low price, workability acidic degradation

reproduction. In the case of skin tissue engineering, it forms a non- to form a solid scaffold with various interconnected pores [68].
homogeneous structure and is limited to the only internal design Soumya et al. [69] have successfully developed an osteoinductive
of the scaffolds. Additionally, the technique is limited to manual herbal scaffold by freeze-drying. They blended medicinal plant
intervention as it involves a time-consuming, multistage process extracts with natural biopolymers [O-carboxymethyl chitosan
that is labor intensive and requires high skill and experience. (CS) and alginate] by lyophilization process. Later, fabricated
Conventional methods do not result in the regularity of pore shape, scaffolds with desirable properties for tissue engineering
reproducibility and sufficient pore interconnectivity [9,49]. applications. The cytocompatibility studies on the developed
composite scaffolds with human mesenchymal stem cells (MSCs)
4.1. Freeze-Drying proved its biocompatible nature. The scaffolds fabricated with
Freeze-drying, also known as lyophilization, is a widely utilized plant extract showed a remarkable difference in cell attachment
fabrication technique. In this technique, it freezes a synthetic and cell reproduction as compared to scaffolds fabricated without
polymer dissolved in selective solvent at a temperature between extract. The developed scaffold showed promising porosity and
−20°C and −80°C, resulting in the solid solvent. The frozen water absorption properties. The freeze-drying technique is mostly
sample then evaporated by sublimation with help of a lyophilizer favorable for biomedical application as water solvents are used
 Kumar and Jacob: Techniques in scaffold fabrication process for tissue engineering applications 2022;10(03):163-176 167

Table 4: Advantages and Disadvantages of different types of scaffolds fabrication techniques for tissue engineering application.
Technique Advantages Disadvantages References

1. Possess higher porosities

1. Process is high energy and time consuming
2. Adjustable scaffolds structure and pore size
Freeze-drying 2. Use of cytotoxic solvents [50]
3. Greater interconnectivity of the porous
3. Leads to shrinkage of the tissue
structure with the extracellular matrix.

1. Use of cytotoxic solvent and leads to the

1. Ease in production denaturation of molecules and cells
Solvent casting and particle 2. Adjustable porosity of the scaffolds and the 2. Bio-inductive property of the molecules decreases.
[51–53]
leaching pore size. 3. Requires a longer time to degrade and a time-
3. High porosity, develops a 3D cell structure consuming process due to the use of thin
membranes
1. Due to the use of organic solvents, they are
non-flammable 1. Process cannot be used as a hydrophilic material
Gas foaming [51]
2. Processing cost is cheap 2. Carbon dioxide used is of low solubility
3. Carbon dioxide is used as porogen gas
1. Porous polymer membrane of anisotropic and 1. Process is not much suitable for seeding of
tubular 3D scaffolds osteoblast cell and maintaining the pore size of the
Thermal-induced phase bone tissue growth.
2. A low probability of defects [51]
separation
3. Low temperature is used for bioactive 2. Use of organic solvents
molecules integration 3. Used for thermoplastic utilization.
1. Can be used in large scale productions
2. Has control over the diameters of the micro 1. Process is limited in producing 3D scaffolds due to
and nanoscale thin fibres poor control over pore structural size and shape
Electrospinning [8,15,54]
3. Abilities to generate homogeneous mixtures 2. Due to the use of the wide range of biomaterials,
with nanoscale fibres the solvents used sometimes might be cytotoxic
4. Develop polymers of high tensile strengths.
1. Requires high operating temperatures where
1. The operating cost of this technique is low.
thermally stable materials can be used and are
2. Process provides high precision and control limited to small pore size
over the microstructure of the complex
SLS 2. Removal of excess trapped material is complex as [12,55,56]
scaffold structure and high porosity
it remains in powdered form.
3. Has great mechanical strength.
3. Post-sinter stage required in this process
4. Post-processing is not required
4. Poor control over surface topography.
1. Provides high accuracy and high resolution.
1. Requires the post-polymerization stage to maintain
2. Forms a complex 3D structure where the strength of the scaffolds
interconnectivity and pores structure maintains
2. Requires photo-polymerization materials of the
uniformity.
Stereolithography low range [12,57]
3. Excess liquid and photopolymer are removed
3. Requires structure support and the abundant
by heating
amount of monomers
4. The cell patterning and growth factors are
4. Uses quite expensive materials.
maintained in this process.
1. Requires a high operating temperature
1. Cost-effective process that has high
mechanical strength and production rate 2. Limited variety of materials range and size of pore
structures
2. Simple process that uses multiple nozzles and
allows moderate temperature deposition of 3. Low utility with non-thermoplastic polymers due
Fused deposition modelling scaffolds to thermal degradation of the polymers [12,58,59,60,61]
3. Solvent-free process. 4. During the process, pre-formed consistent-sized
fibres are needed to feed through rollers and nozzle
4. High porosity where the structure and size of
and are limited for the application in biodegradable
the pores can be adjusted
polymers [58].
1. Material is quite expensive
Multiphase jet solidification 1. Performs contiguous high resolution [62,63]
2. A good rheology control
1. Used for the fabrication of high scale precision
Precise extrusion deposition micro and complex scaffold 1. Requires high temperature [62]
2. Materials used are in the form of a pellet
1. The pore size of the scaffold are well
1. The scaffold developed have low mechanical
connected
3D Bioplotting strength [51,62]
2. Use of abundant amount of biomaterials and
2. Difficult in operating due to slow speed
biomolecules
1. A high-speed tractable o droplet size precision
process
1. Lacks in the precision of droplet deployment and
2. Vast amount of biomaterial are available adequate size
Inkjet printing [14,64,65]
3. A low-cost process with high resolution, high- 2. It requires low viscosity for the bio-ink.
throughput capability, reproducibility, and easy
to use
168 Kumar and Jacob: Journal of Applied Biology & Biotechnology 2022;10(03):163-176

Figure 1: Conventional methods of scaffold fabrication [66,67].

rather than other organic solvents for the fabrication of scaffolds polylactic acid (PLA) scaffold with high porosity using methods
but remain challenging for the fabrication of classified structured like phase separation, particle leaching techniques, vacuum-
scaffold-like vascular systems used in biomedicine applications assisted solvent casting, and solvent extraction. In this process,
[70]. The cytotoxic solvents used for the mixing of the polymers the surface is modified with the help of a CS/osteogenic growth
consume high energy. The developed scaffold requires washing peptide (OGP) coating layer that can be potentially applied in
several times to remove the toxic solvents and hence declines cell bone, cartilage regeneration and tissue engineering purposes.
death as well as triggers the irregularities in pore sizes for a longer The experiment proved that the higher the porosity of the PLA
time- span [71,72]. scaffolds developed, the lower the PLA solution concentration and
the temperature. The hydrophilicity and mechanical properties of
4.2. Solvent Casting and Particle Leaching CS/OGP/PLA developed scaffold were higher as compared to the
uncoated PLA scaffold.
This method is generally used for Bone and cartilage tissue
engineering applications [73]. In this technique, a highly volatile
solvent solvates the polymer and cast in molds along with 4.3. Gas Foaming
porogen. The solvents evaporate with a matrix consisting of salt Gas foaming utilizes natural cytostatic solvents and high
particles and the formed composite matrix contains porogen and temperature. In this method, the fabrication model uses inert gas
polymers. The matrix submerged in water allows salt leaching foaming agents like methane, carbon dioxide, hydrogen, nitrogen
to develop scaffolds structure with high porosity. The solvent to pressurize with a biodegradable polymer like fluoroform or
combined with uniformly distributed organic amalgams like water solvents until it reaches saturated conditions to form gas
glucose, gelatine microsphere and otherwise soluble inorganic bubbles [76]. The gas foaming techniques and fused deposition
salts like potassium chloride is selectively leached to get a certain methods are combined where, the porous PLA scaffolds dominate
pore size which is used as porogen to dissolve in the polymer 1 to 10 μm micro pores sizes structures. Gas foaming process
solution [74]. The scaffolds prepared through this technique have generates micropores (˂10 μm) while in conventional 3D process,
a porosity of 50% to 90% and are of a low-cost process [75]. it is barely developed. The fused deposition method incorporates
Zeng et al. [66] successfully fabricated and developed 3D porous macro porosity through attached channels that have developed the
 Kumar and Jacob: Techniques in scaffold fabrication process for tissue engineering applications 2022;10(03):163-176 169

saturation capabilities by reducing the time consumptions in the exhibit great porosity, high surface area, and biomimetic like
process. This technique achieves generally 85% porosity and 30 natural extracellular matrix. During this process, the polymeric
to 700 μm pore size for the developed structure which is sponge droplet is executed by the stress at the needle tip by using an
in nature [58]. Song et al. [67] developed tailored macro/micro- electrically charged jet. Then at high voltage, the charging solvent
porosity architectures scaffolds by combined technology of gas gets dominated by an interaction between electrostatic repulsion
foaming and fused deposition modelling process for applications and surface tension, where the spinneret droplets erupt and gets
in tissue engineering technology. stretched by passing through grounded collector from the spinneret
tip. Finally, the jet solidified into nanofibers as the solvent starts
The PLA is blended with poly(vinyl alcohol) (PVA) to fabricate
evaporating [80]. For the production of nanofibers, various
composite filaments by fused deposition modelling. After the
parameters are followed such as surface tension, conductivity,
fabrication process, the developed scaffolds were dominated by the
flight distance, type and concentration of polymeric solvents,
gas foaming process to create micropores of size less than 10 µm.
viscosity, spinneret diameter, interactions between the molecules,
The outcomes of this process revealed that without further dense
the electric current supplied, rate of flow, types of collector [81].
skin layers, interconnected pores attained micropores size of 2 to
Yuan et al. [82] has successfully developed nanofibrous scaffolds
10 µm. Hence the scaffolds developed have great potentialities for
with polyethylene oxide (PEO) and CS using the electrospinning
cartilage and bone tissue regeneration. Manavitehrani et al. [77]
fabrication technique. In this study, with the reduction of mass
have studied polyester-based poly propylene carbonate (PPC)-
of CS and PEO from the scaffolds, degradation is exhibited. The
starch bioscaffolds in tissue engineering technologies. In this
bactericidal study shows stronger growth inhibition and cytotoxic
process, to develop a porous scaffold, the bi-products are degraded
effects. This fabrication technique is categorized into three
and fabricated by gas-foaming technique with PPC blended with
depending on the types of manufacturing methods consisting
starch and bioglass particles. The pore sizes developed were
of solution materials by changing electrospinning materials,
ranged from 100 to 500 μm, with high pore interconnectivity.
setting up of collector by using liquid-assisted collectors with
perfunctory setup, post-processing operation after electrospinning
4.4. Thermal-Induced Phase Separation (TIPS) [8]. Hejazi and Mirzadeh [83] have developed 3D scaffold with
In the TIPS process, various polymeric solutions of solvent and electrospraying and electrospinning combined technique by using
non-solvent are demixed, quenched and consist of different polycaprolactone (PCL). The 3D scaffold was fabricated using
polymeric phases. This demixing process takes place either by macro and nanofibres particle with optimized pore size, porosity,
evaporation or extraction process, solvent is removed resulting interconnectivity within the pores and biomimetic the extracellular
in pores formation [74]. In this technique, the mixing of various matrix structure. Although electrospinning is widely used, there is
types of selective solvent, additive blenders, and multicomponent various limitation such as distribution of sufficient homogeneous
polymer are used at low temperature for developing force pores sizes, limited to some applications in biomedicine [70], and
separation process, where the homogeneous polymer solution the solvent used is toxic and depended on various variables.
at high-temperature environment settled down to induce phase
separation by achieving variant polymeric phases [75,58]. The 5. RAPID PROTOTYPING (RP)
scaffold structures are attained, as the solvent gets eliminated by
Due to various limitations in the conventional methods, the
the freeze-drying process with relatively porous and nano-scale
use of RP technique was introduced to develop 3D porous
fibrous meshes. The thermoplastic crystalline polymer scaffolds
scaffolds with great architecture advantages, higher porosity
are generally used for construction and low temperature is mostly
and interconnectivity within the pores. This process, also known
used for blending bioactive materials with the fibrous scaffold [9].
solid free-form fabrication technique, is a strong fabricating tool
In this method, the porosity of the fibres is achieved 98% higher
and immensely used for the preparation of scaffolds in tissue
than the surface to volume ratio of the scaffold constructed. Biswas
engineering. The scaffolds are developed by using computer-aided
et al. [78] have developed porous CS scaffold by using combined
designing tools to provide a perfect fit architectural structure with
technology with mechanical foaming and thermal-induced phase
physio-chemical properties. In the RP process, closely attached
separation technique and obtained 80% porosity, 2.6 to 25 kPa
materials are combined with powder, sheet or liquid and the
adjustable compacting parameters with 120 mm pore size. The
fabrication process is employed by the addition of consecutive
scaffold showed great potential for tissue engineering and the
layers to produce 3D scaffolds utilizing the computer-generated
foaming process incorporated by air bubbles, functioned as a
models [84]. Initially, development takes place for a 3D volumetric
mould for the macro-porous construct of the developed scaffold.
computer model which is derived from yield information produced
In this technique, materials are limited to the fabrication process,
by surface digitizers or by clinical imaging frameworks. Later, the
inadequate resolution and very selective minimal materials
digital model extracted is stacked and fused on top of each other
are used in the phase separation process with uniform porous
to make user-defined structures that statistically cut into layers
structures [70].
with a consistent thickness [49]. The main advancement of these
fabrication techniques is that they can maintain the porosity, pores
4.5. Electrospinning shape and size of the scaffolds and have highly interconnected
Electrospinning is extensively used in nanofibers polymers and for pore structure. It additionally empowers for development of
scaffolds fabrication process from the selective solution by using patient-specific customizable scaffolds that are appropriate for
electric current [79]. In this technique, the nanofiber scaffolds tissues and organs designing technology [58]. There are various
170 Kumar and Jacob: Journal of Applied Biology & Biotechnology 2022;10(03):163-176

Figure 2: Types of RP fabrication techniques for tissue engineering [85].

types of RP fabrication techniques for tissue engineering purposes scaffolds by using the SLS technique. In the cycle, four distinctive
(Fig. 2) such as fused deposition modelling, stereolithography, composite powders with about 75% (wt%) polylactide (PLLA
electron beam melting, 3D printing and SLS [9]. and poly(D,L-lactic acid) and 25% (wt%) calcium carbonate
(calcite) composite were set up by milling process dependent on
5.1. Selective Laser Sintering (SLS) Good Manufacturing Practice principles, where the four different
grades of polylactide were selected for shelling the broad inherent
SLS is a powder-based fabrication technique that uses laser
viscosity range of 1.0–3.6 dl/g. The composite material with the
technology to sinter powdered material such as polymers, ceramics,
most minimal inherent viscosity at (1.0 dl/g) showed the best
and metals. It is utilized for manufacturing and developing the
processability by SLS process where the biaxial twisting quality of
scaffolds for fabrication in tissue engineering [9]. Patient-specific
up to 75 MPa was accomplished. While the cell culture measures
implants are developed with composite interconnectivity pore
showed great feasibility of MG-63 osteoblast-like cells on the SLS
structures to advance bone ingrowth [86]. Although there are
exhibits. At last, the 3D scaffolds with great pore structure and
various process for the treatment and developing solutions to treat
interconnectivity with the pores were developed [87].
bone imperfections such as with titanium and polyetherketone
ketone inserts as they are non-degradable but have the chance of
getting several complications in future [85]. The productivity of 5.2. Stereolithography
this method is that it provides outstanding dominance over the Stereolithography technique is the process of developing solid 3D
microstructures of the developed scaffolds under various parameters scaffolds based on structurally regulated solidification of the fluid
like compositions of percentage by physically mixed polymers or resin by photo-polymerization measure. In this process, the printing
by composite powder mixes to get the properties of the favoured of thin layers of ultraviolet curable material is done layer by layer
scaffold [87]. Gayer et al. [85] have successfully developed and using photolithography pattern [88]. The four main components in
fabricated solvent-free polylactide-calcium carbonate composite this process are UV laser for radiating resin, photosensitive liquid
 Kumar and Jacob: Techniques in scaffold fabrication process for tissue engineering applications 2022;10(03):163-176 171

resin, transferable built platform, and dynamic mirror system [9]. controlled cross-hatch microarchitecture depending on various
The process starts by depositing the surface layer of photosensitive printing parameters like printing temperature, infill angle, and
liquid resin with a UV laser and solidified to a characterized depth. layer thickness. It is then adjusted directly along with the use
of readily available filaments and can be used in developing
The initial layer is solidified and the platform is lowered pharmacological products of good auxiliary properties with the
down vertically layer by layer through photo-polymerization least post-processing requirements. Kalita et al. [94] developed and
methods. The assisted platform is moved from the surface and fabricated 3D particulate-reinforced polymer-ceramic composites
the assembled layer is recoated with fluid resin. At that point, of polypropylene (PP) polymer and tricalcium phosphate (TCP)
the subsequent layer is put over the principal layer where the ceramic by high shear blending process with adjusted porosity.
process persistent until the 3D scaffold is created. Lastly, the The PP-TCP composite fibers are prepared to utilize a solitary
uncured sap wiped out and the scaffolds are post cured under screw extruder followed by fused deposition to manufacture
UV light with controlled exposure time, light intensity [9]. permeable structures. In this process, the strength of the composite
Farzan et al. [89] developed an elastic 3D-printed scaffold by was decreased as pore volume increased to identify through
stereolithography and solvent-free methods followed for the compression testing. The porous scaffolds developed were later
fabrication process using PCL and polyethylene glycol (PEG). characterized to use as bone grafts. Chen et al. [95] manufactured
The outcomes demonstrated that PEG-containing PUs had higher PVA/β-TCP composite scaffolds with a combination of solid-state
degradation rates, and the elasticity of PU/PCL/PEG was 1.4 and shear milling and fused deposition methods. For the development
twice higher than that of PU/PEG and PU/PCL, separately. The of bioactivity and osteo-conductive properties, β-TCP bioceramic
3D printed PU/PCL/PEG demonstrated high appropriateness material is used as the fortifying filler while PVA as the polymer
in delicate tissue engineering during the scaffold development grid. The composite scaffolds formed demonstrated that β-TCP
process. Elomaa et al. [90] developed photo-cross linkable PCL particles homogeneously scattered into the PVA grid with the help
based resin utilizing solvent-free stereolithography methods. In of solid-state shear processing. The outcomes firmly demonstrate
this process, the photo-cross linkable macromers was set up by the capability of the fabricated scaffolds in tissue engineering
methacrylating three-outfitted oligomers alongside methacrylic applications. The deposition takes place at low temperature
anhydride where the porous scaffolds were developed by though encapsulation of cells during the fabrication process was
stereolithography technique using PCL resin. The ideal resin not allowed [59].
viscosity was formed during the curing process by heating the
resin. Due to absence of solvent, the scaffolds developed provide
6.2. Multiphase Jet Solidification
the best fits with CADs as the material shrinkage do not take
place. The interconnectivity of the pores was high in the photo- In this technique, high thickness metallic and ceramics parts are
cross linkable biodegradable PCL resin and has a great potential created by utilizing low melting point compounds or powdered
for tissue engineering scaffolds [91]. blender by compressing out through computational controlled
spout to fabricate the part layer-by-layer [49]. The very important
parts in this process are controlled with computational positioning
6. EXTRUSION-BASED SYSTEM
arrangements and a warmed chamber with a stream and a pulling
Extrusion based techniques are the advanced fabrication technique framework. The materials in this technique are stacked in the
because of various advantages like high resolution, drug-loading, structure of material-binder mixture, powder, bar, and pellets
cell-friendly environments, high physical properties, feasibility which are heated in a reservoir system or by the processed
controlled over drop-on-demand high precision deposition [92]. chambers exceeding the melting point of the binder. Gradually
This technique is mechanically accessible and are less expensive throughout the process, the binder gets liquefied where the heated
with correlation to other solid freeform manufacturing process. paste is propelled out through a heated jet spout and settled onto a
The extrusion-based system depends on material melting where controlled computational system [59]. Xiong et al. [96] developed
computer-aided designing software is used and scaffold is formed and fabricated porous PLLA/TCP scaffolds through a computer-
layer after layer. This technique is categorized based on material aided low-temperature deposition manufacturing process for bone
melting methods and type, however, the heat method is commonly tissue engineering applications. The developed scaffolds showed
used, some of the other methods like premixed pastes and inks great porosity of 89.6% besides that scaffolds has controlled
methods are also used [93]. The various process of the extrusion- interconnectivity pores, great biocompatibility, great bone
based system includes fused deposition modelling, multiphase jet conductivity, and suitable in vivo biodegradable property which
solidification, precise extrusion manufacturing, and 3D plotting. make them suitable for tissue engineering.

6.1. Fused Deposition Modelling 6.3. Precise Extrusion Deposition

It is a hot melting extrusion process wherein, the casting of the The Precise Extrusion Deposition technique developed scaffolds
solid polymer takes place through a nozzle which is ejected and with controlled structural adaptations and pore sizes that can
melted on the surface of 3D patterns by utilizing a controlled provide strength, structural integrity, and microenvironment for
computer-aided tool for extrusion and deposition process [9]. In tissue regeneration and tissue engineering [97]. This method is
this process, the 3D scaffold is made up of layer-by-layer method, more applicable in the pharmaceutical field. PCL, one of the most
where multiple layers of adjacent microfilaments take places. widely used biodegradable polyester has a low melting point and
In this technique, the tailoring of bone scaffolds is done with are approved by the Food and Drug Administration with peculiar
172 Kumar and Jacob: Journal of Applied Biology & Biotechnology 2022;10(03):163-176

applications which are particularly utilized for longer run embeds jet dispensing process where ink droplets or liquid material
and controlled drug delivery applications [97]. The precision are discharge into the substrate with predetermined patterns
extruding deposition process is the variations of fused deposition [88,97]. The liquid material is forced through the nozzle by
modelling technique, where without filament preparation the pressurizing the stream of droplets to form the bio-ink solution
scaffolding material are directly deposited [98,99]. Shor et al. [100] which is then made up for cell culture medium or the hydrogel
has developed scaffolds using a precision extrusion deposition [104,64]. In this technique, print heads are provided by the
process by fabricating PCL-hydroxyapatite composite through printer that is connected to nozzles through ink chambers.
computer-aided tissue engineering technique. In this study, the The droplets stipulated are signaled in the form of the pulse
capability of the precision extrusion deposition fabrication process from the electrostatic, thermal, and piezoelectric actuators
of the scaffolds with structural integrity, controlled microstructure, which produce pressure in the liquid materials [104]. While
pore interconnectivity pore size, mechanical property is required the surface tension of liquid material gets surpass by the
for cartilage and bone tissue engineering have been characterized. pressure formed in the nozzle orifice and finally ink droplet gets
The developed scaffolds formed 60%–70% porous and with 100% emitted out through the nozzle tip. Later through the nozzle,
interconnectivity. This technique presented great advantages for high voltage is applied to the generated substrate. The liquid
high-precision on the micro-scale for complex scaffold fabrication material gets encircled by the meniscus which prises the ink
as compared to the conventional fabrication of scaffold methods. with the substrate where the electrical power is constrained by
surface pressure, that deposit on the substrate [97]. Generally,
6.4. 3D Bioplotting there are two different methods for this type of technique
i.e. piezoelectric-actuated ink jet printing and thermal inkjet
3D Bioplotting is one of the versatile and widely used techniques.
printing. Zamanifard et al. [105] have developed and fabricated
It involves the mapping of a 3D moving head extruder which
natural polyhydroxybutyrate biocompatible polymer scaffolds
is controlled by the use of packed air to compel out fluid or
by using electro-spun technology and computer-aided software.
glue-like plotting medium to produce 3D solid scaffolds. Then
The modelling of scaffolds was based on data received from
the characterization is done based on different compositions
the software with response to the exterior and artificial neural
of material such as tailor-made internal structures, pore
matrix strategies. The various data generated are compared
interconnectivity, and complex shapes. The scaffolds developed
with experimental results. In this inkjet process, the natural
in this technique are fabricated layer-by-layer, where the plotting
polymer polyhydroxybutyrate was electrospun and bioscaffolds
materials are stored in the form of a movable dispenser. Then
with high biocompatibility were developed of 224 to 360 nm
with the help of a heating jacket and with nozzle, the gaseous
breadth range. However, the final results confirmed that the
tension is controlled into fluid plotting medium are plotted and
scaffold developed by printing polyaniline nanoparticles and
maintained [59]. In this process, the polarity and density of the
oxygen plasma methods with defined designs has a great effect
fluid medium are in a limited manner along with plotting material
on cell attachment and cell development. In addition, was non-
for prevention of gravity-induced structural collapse and to get
harmful for human fibroblasts and is reasonable for considering
temporary support structures [101]. Naghieh et al. [102] have
the impact of electrical incitement on human fibroblasts. The
developed alginate scaffolds by an indirect-bioprinting process
developed scaffold exhibits various uses in nerve and tissue
that is categorized for nerve tissue engineering applications.
engineering technologies established by the degradation studies.
These low-concentration alginate scaffolds are developed
through the indirect-bioprinting process. It involves various
methods that include printing a conciliatory system from 6.6. CAD Technology
gelatin, impregnating the structure with low focused alginate, The CAD technology makes scaffold fabrication a cheap, safe
and eliminating the gelatin system by incubation process, and and time convenient by replacing conventional drawing board
shaping low-fixation alginate scaffolds. The mechanical and methods. The CAD process uses multidimensional coordinating
biological properties of the developed scaffolds are influenced system where three-dimensional data are generated digitally with
by the accumulation of alginate and the disinfection method the uses of noncontact 3D laser scanner and rapid-measuring
used which give a viable method of adjusting scaffold properties technology. It is then viewed in broad array of perspectives
during the backhanded bioprinting measures. Gómez-Lizárraga and results are procured with the uses of RP process. The CAD
et al. [103] have developed a 3D bioplotting process for scaffolds based scaffolds (Table 5) are the advanced integrated fabrication
fabrication which is made up of a composite of PCL and ceramic techniques. The computer-aided designing software include
micro-powder. The scaffold was manufactured in a cell grid AutoCAD [116], FreeForm Plus software [117], MathMod,
structure. The scaffold developed had a porosity of 32% and a Meshmixer, Netfabb, and Cura [107] and used libraries such
pore size of 323 μm and that indicated a potential use in tissue as Visualization Toolkit (VTK), numpy and wxPython libraries
regeneration and tissue engineering applications. that provide complexity geometric primitives [118]. AutoCAD
(Autodesk) is a specialized CAD application to develop 2D and
6.5. Inkjet Printing 3D model precisely with respect to its dimensions from micro to
macro structures and arrangements with the pores sizes. Similarly
This technique is also known as drop-on-demand or electro-
poncad [119], meccad [120], solidwork [121] and blockscad
dynamic inkjet printing. It is a non-contact technique that
[122] are associated with designing of scaffolds. Ansys Fluent
is controlled by the physical properties depending on the
[123] is to visualize the scaffold with respect to fluidic properties
 Kumar and Jacob: Techniques in scaffold fabrication process for tissue engineering applications 2022;10(03):163-176 173

Table 5: List of Software used in Scaffold design and fabrication.

Software used and purpose Method of fabrication Material Application Reference
Abaqus assembly
Fused filament Soft biological
Module: geometrical files in PCL [106]
fabrication tissue scaffolds
stereolithography format were exported
MathMod: to visualize and animate parametric
surfaces.
Mesh- mixer: a3D design software to change
porosity of structure. Extrusion-based 3D Bone tissue
PLA, [107]
printing, engineering
Netfabb: to measure porosity
Cura: to change the dimensions of the whole
structure.
SOLIDWORKS 17.0: for initiation of geometry was Titanium dioxide (TiO2)
initiated. Powder metallurgy and Alumina (Al2O3)
Femur bone [108]
finite element ANSYS process nanoceramic
15.0 software programs for modeling and analysis. particles
Ceramic bone
CAD to obtain Geometry suitable for fabrication Direct ink writing Ceramic hydroxyapatite [109]
scaffolds
Mathmod (V3.1) software: to generate files to
describe the surface of Gyroid. Electron beam melting Ti-6Al-4V gyroid Bone implant
[110]
Rhinoceros, netfabb software: for scaling to create (EBM) scaffolds applications
various unit cell sizes and final model preparation.
Of poly(L-lactic-co-
CAD and finite element analysis for designing.
glycolic acid), type I Bone tissue
COMSOL Multiphysics software: to validate CAD 3D bioplotting. [111]
collagen, and nano- engineering
scaffold design.
hydroxyapatite
Powder-based three- A high-performance
CAD system 3D design software SolidWorks®2012 dimensional printing Bone tissue engi-
composite material [112]
and exported as an STL file neering
stere-olithography (Zp150)
Polymer ABS material
Fused deposition Bone tissue
MakerBot Replicator 2 FDM modeler for RP poly(lactic-co-glycolic [113]
modeling engineering
acid)
SolidWorks software to create scaffold patterns. Robocating or direct ink
Hydroxyapatite (HA) HA bone scaffolds [114]
ABAQUS/CAE software: for FE simulations writing,
PCL Bone tissue
RPTools software for RP SLS [115]
4% HA engineering

and Abaqus [124] applied in modelling and finite element drug delivery, bone repairing, tissue-related various engineering
analysis. VTK [125], is an open-source, freely available software processes.
system for visualization, processing of the image and developing
three dimensional computer graphics. wxWidgets/ wxPython 8. CONFLICTS OF INTEREST
[126] is a freely available program that allows to develop highly The authors report no financial or any other conflicts of interest
graphical user interfaces. NumPy [127] is specialized program in
in this work.
data analysis and numerical calculation for large dimensions of
data using array processing Python package. 9. PUBLISHER’S NOTE
7. CONCLUSION This journal remains neutral with regard to jurisdictional claims
7. CONFLICTS OF INTEREST
Tissue engineering is a vast multidiscipline area with a wide range in published institutional affiliation.
Theapplications.
of authors report
Thenofabrication
financial orofany other conflicts
scaffolds is a veryofcomplex,
interest
dedicated, and sensitive process due to its various factors and
parameters. Based on literature surveys, it can conclude that the RP
techniques as an advanced fabrication technique that uses computer
aided software and tools for scaffold development in tissue
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