3D Printing and/or 3D Bioprinting technique(s)

 3D Printing and/or 3D bioprinting technique(s):

 Biomaterials (cellulose, alginate, collagen -hydrogels- ) are used as bio inks to make scaffolds.
 The main 3D printing techniques are: (1) Extrusion based 3D printing (Fused Deposition
Modeling (FDM) a.k.a Fused Filament Fabrication (FFF), and the latter is known as Robucasting
or Direct Ink Writing (DIW) a.k.a Liquid Deposition Modeling (LDM)); (2) Photo-
polymerization based 3D printing (Stereolithography (SLA) - laser based SLA printers - or vat
polymerization), and another technology of vat polymerization is Digital Light Processing (DLP)
or DLP printers); (3) Powder based 3D printing (employ a laser beam or a chemical binder to
locally bind the powder materials layer by layer on powder bed, binder jetting method,
Selective Laser Sintering/Selective Laser Melting (SLS/SLM)).
 Fabrication: the scaffolds needs to be biocompatible, biodegradable, mechanically compatible
and bioactive.
 Tissue Engineering: is a promising field of regenerative medicine that relies on the interaction of
three elements: a supporting material, growth factors, and cells to develop a biological substitute
for the replacement, restoration, or regeneration of damaged tissues and organs. Intini2018
 The challenge of this matter is to mimic what happens in nature.
 Biocompatibility and proangiogenic properties: can be evaluated using HE, Masson, CD31 +,
and collagen type III immunohistochemical staining.
 The production of ECM: plays a pivotal role in the regeneration process, driving cell
proliferation, differentiation, and maturation.
 Bio-fabrication and use of porous 3D scaffolds as appropriate environments for cell colonization
and proliferation, thereby enabling the production of ECM and the reconstruction of complex
tissues (to create scaffolds with arbitrary geometries and heterogeneous material properties,
which can mimic the complexity of native tissues.
 Chitosan and chitin present haemostatic action which can be exploited to enhance healing
process. Intini2018 Chitosan doesn’t elicit/generate adverse reactions in contact with human cells,
is not allergenic and is widely available as bio waste product of seafood processing industry.
 ECM (Extracellular matrix): is an intricate network composed of an array of multidomain
macromolecules organized in a cell/tissue-specific manner. Components of the ECM link
together to form a structurally stable composite, contributing to the mechanical properties of
tissues. The ECM is also a reservoir of growth factors and bioactive molecules. It is a highly
dynamic entity that is of the ECM link together to form a structurally stable composite,
contributing to the mechanical properties of tissues. The ECM is also a reservoir of growth
factors and bioactive molecules. It is a highly dynamic entity that is of vital importance,
determining and controlling the most fundamental behaviors and characteristics of cells
such as proliferation, adhesion, migration, polarity, differentiation, and apoptosis.
 The extracellular matrix (ECM) function : helps cells to bind together and regulates a
number of cellular functions, such as adhesion, migration, proliferation, and differentiation.
It is formed by macromolecules, locally secreted by resident cells. The two main classes of
macromolecules are polysaccharide glycosaminoglycans, usually covalently linked to proteins
in the form of proteoglycans, and fibrous proteins of two functional types, structural (collagen,
elastin) and adhesive (fibronectin, laminin, vitronectin, etc.).
 ECM-derived hydrogels offer great promise as biomaterials for tissue engineering as they
can be delivered to the site in need in a minimally invasive manner, allow cell encapsulation
prior to delivery, but can also allow for neighbouring cell recruitment. ECM hydrogel
solutions have also been used as a bioink for 3D bioprinting several different tissues and organs.
 One of the main benefits of using ECM-derived hydrogels is the ability to retain multiple
ECM components that may not be present in other natural or synthetic hydrogels and
therefore more closely mimics the native tissues.
 The hydrogels will need to be tested in vivo to evaluate the immunological response and
ability to integrate with the surrounding tissue. The survival and functionality of the hydrogels
would also need to be monitored. Should the hydrogel not induce any negative effects in vivo
then the potential for hydrogels to replace damaged or diseased tissue could be explored using
suitable animal models.
 Each organ and tissue is composed of a distinctive ECM, in its biochemical composition and
structural organization. The properties of ECM are important in the fields of tissue
engineering and regenerative medicine, which often aim to replicate the composition and
structure of the ECM. By using synthetic or natural materials, three-dimensional (3D)
scaffolds can be fabricated to repair or restore damaged organs and tissues.
 Decellularization is the process of removing the cellular components of a scaffold while
retaining the macrostructure and microstructure of the ECM. Various methods may be used
for this process and may involve a blend of chemical, physical, and enzymatic treatments.
Decellularization results in a decellularized matrix also referred to as acellular or devitalized
matrix in the literature.
 The decellularized scaffold is beneficial as it retains the three-dimensional structure of the
parent tissue and provides a precise microenvironment required for the seeded cells to
thrive, differentiate and function. Since the composition of the decellularized scaffold
simulates the native tissue, the regenerative cues are inherently available in the scaffold which
considerably minimize the adverse immune responses in the host body following implantation. 
 Decellularization process aims to remove all cellular and nuclear matter minimizing any
adverse effects on the composition, biological activity, and mechanical integrity of the
remaining ECM for the development of a new tissue. The process usually consists of
mechanical shaking, chemical surfactant treatment, and enzymatic digestion.
 Tissue decellularization (method): one popular approach to generating scaffolds that try to
imitate the tissues or organs ECM characteristics is to use decellularization. This technique
involves the removal of cellular components from a tissue so that only the ECM remains. Many
methods have been examined for performing decellularization and these can be divided into three
main categories: physical, chemical and biological. Physical methods include freeze-thawing
cycles, high hydrostatic pressure or supercritical CO 2. Chemical agents can involve ionic
detergents, such as sodium dodecyl sulphate (SDS) or sodium deoxycholate; non-ionic
detergents, such as Triton X-100; hypertonic or hypotonic salt solutions, such as sodium chloride;
and acids and bases, such as peracetic acid or ammonium hydroxide. Enzymes such as trypsin,
dispase and phospholipase A2 have been used as biological methods for decellularization.
Furthermore, nucleases, such as DNAse, are used to promote the fragmentation of residual DNA
into <200 bp fragments in order to minimize immunological responses. Extensive research has
been completed to optimize these decellularization procedures to allow for maximal cell removal
and minimal ECM damage for each tissue/organ.
 Fabricating scaffolds for tissue regeneration using synthetic and natural materials. In contrast to
large lot variation, low mechanical strength and uncontrollable degradation of natural materials
(e.g., collagen, chondroitin and hyaluronic acid), synthetic polymers offer better control of the
physicochemical properties and processability. However, oftentimes the synthetic polymers may
elicit unwanted inflammation and lead to undesirable tissue formation as a result of acidic
degradation by-products. In recognization, increasing efforts also shift to develop ECM scaffolds
by decellularizing and cultured cells or native tissues.
 Physicochemical properties, e.g., pore size, porosity, bio activities, stiffness, etc.
 Rejection of scaffolds and desired mechanical properties are still challenging e.g., immune
rejection (requires to minimized immune responses).
 Numerous fabrication methods have been developed to tailor tissue engineering (TE) of scaffolds
to fulfill diverse needs.
 Physicochemical characterizations of scaffolds: SEM, XRD, FTIR, XPS, TEM, EDX
 SEM and TEM: are used to explore the architectural features of the scaffolds (to reveal an
interconnected nano-fibrous network structure – is it similar to they native ECM? –
 Enzymatic degradation: in vitro degradation of the scaffolds can be carried out using lipase
enzyme at concentration of 10 units/mL.
 In vitro degradation properties: evaluated/measured by the weight loss rate of materials. (e.g.,
observed after 14 days of degradation).
 The mechanical properties of dermal substitutes must be appropriate for clinical applications.
An ideal dermal scaffold should have a suitable elastic strength (modulus of elasticity/Young’s
modulus and elongation at break), and should be able to maintain a complete shape in the initial
stages of wound repair. Stress-strain curves: the fracture stress (Mpa) and the fracture strain (%).
Good scaffolds elasticity demonstrate with the fracture strain greater than 10%.
(10.1016@j.cej.2020.128146) E=stress/strain (Young’s modulus is determined by the
relevant stress-strain curves)
 Antibacterial properties (antibacterial activity): is characterized by soft agar inoculated with
bacteria and liquid tests to study bacterial growth inhibition and bactericidal action respectively.
It can be tested by using Staphylococcus epidermidis (S. epidermidis which is a Gram positive
bacteria) and/or Escherichia coli (E. coli which is a Gram negative bacteria) for instance.
 Bacterial growth inhibition test: to assess the ability of the material to prevent the implanted
site from the formation of bacterial biofilm in presence of nutrients since it could lead to bacterial
infection. It is tested by using soft agar. Negative control plates (i.e. without bacteria and
scaffold) and positive control plates (i.e. with bacteria and without scaffold) + (soft agar with
bacteria and scaffold).
 Bactericidal activity: to assess the ability of the material to remove bacteria that has already
grown on the material implantation zone. Bactericidal activity was assessed by liquid tests in
which the material (90 ± 10 mg) was immersed in a bacterial suspension (10 mL) at about 10 2
CFU/mL. The associated log-removal values, where the log-removal is defined as the logarithm
(log) ratio of the bacterial concentration C measured after respective contact time relative to the
initial bacterial concentration C 0. A log-removal value of -log (C0) was attributed to the particular
case of total removal of the cultivable bacteria. A log-removal value higher than 1 is related to a
significant bactericidal activity.
 In vivo: in vivo is tested to evaluate the immunological response and ability to integrate with the
surrounding tissue (in vivo validation of scaffold performance).
 In vivo: is a Latin for “within the living. It refers to work that’s performed in a whole, living
organism.
 In vitro: “within the glass. When something is performed in vitro, it happens outside of a living
organism.
 In situ: “in its original place”. It lies between in vivo and in vitro. Something that’s performed in
situ means that it’s observed in its natural context, but outside of a living organism.
 Hydrophilic nature (hydrophilic surface): the contact angle between surface and water droplet
is less than 90°, as a result when the water droplet falls in the surface of the material, it will be
adsorbed on the surface of the material instead of rolling off.
 Hydrophobic nature (hydrophobic surface): the contact angle between surface and droplet is
greater than 90°, then such a surface is known as the hydrophobic surface. For a super
hydrophobic surface, the contact angle should be greater than 150°, in such a case water droplet
attains spherical shape and roll off the surface.
 Skin tissue: It is our first line of defense against many types of infections deceases, but it is also
susceptible to damage from injuries like burns and cuts. When it is not treated immediately, these
injuries can be infected and very severe injuries require skin grafts. But, a close match is required
 between donor and host response to accomplish that (it should be able to mimic the nature of
organs).
 Skin: is the first innate immune barrier that protects human body from internal aggressors. It is
easily damaged by various factors, such as burns, accidents, and diseases.
(10.1016@j.cej.2020.128146)
 Tissue regeneration requires rapid vascularization (angiogenesis) in order to provide nutrients
to support the survival of relevant cells and maintain normal cellular functions.
 Vascularization:
 Dermalization:
 The generation of vascular networks is essential for the prevention of hypoxia, apoptosis, and
tissue necrosis. The speed, success, and quality of the regenerative repair process of damaged
wounds depends on the degree of early vascularization.
 Artificial dermal substitutes are often referred to as “artificial dermis layers” which is 3D
scaffolds that are made up of natural or synthetic materials designed to enhance adhesion and
growth of dermal fibroblasts, especially during the treatment of deep wounds.
 Fibroblasts:
 Proliferation: cell proliferation activity is evaluated using the CCK-8 cell counting method.
Cell proliferation is shown by the movement of many green cells along the fiber surface of the
printed scaffold. “Proliferation/spread/breeding/dispersion”
 Proliferative ability
 Phalloidin staining: is a highly selective bicyclic peptide that his used for staining actin
filaments (also known as F-actin). Visualizes actin in cells and tissues.
 DAPI staining: visualizes nuclear DNA in both living and fixed cells. DAPI staining is used to
determine nuclei and to assess gross cell morphology. DAPI staining allows multiple use of cells
eliminating the need for duplicate samples.
 Adhesion:
 Migration:
 Polarity:
 Differentiation:
 Apoptosis:
 Cytocompatibility: The cell proliferation. All the scaffolds promoted the adhesion and
proliferation of fibroblasts. Fibroblasts that adhered to the surface of scaffolds were observed
after live/dead staining.