Page 1
ASSIGNMENT -I
DIFFUSION BASED AND VASCULAR CONSTRUCTS,
TRANSPORT OF NUTRIENTS AND METABOLITES
18/04/2016
Dr. Prabha D. Nair
Scientist 'G' (Senior Grade) & Scientist-in-Charge
Tissue Engineering and Regeneration Technologies Division
By
YANAMALA VIJAY RAJ
MTECH IN CLINICAL ENG
BT14M004
Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum
Page 2
CONTENT
1 TISSUE
ENGINEERING…………………………………………………………………………………1
1.1 PROCEDURE FOR TISSUE ENGINEERED PRODUCT …………………………………1
2. STRUCTURE OF BLOOD VESSELS ……….……………………………………………2
2.1 TUNICA INTIMA …………...………………………………………………………………3
2.2 TUNICA MEDIA ……………………………………………………………………………3
2.3 TUNICA ADVENTITIA ………………………………….…………….……………………4
3. BLOOD VESSEL FORMATION ………………………………….……….……………4-5
3.1 ENDOTHELIAL CELLS ……………………………………………………………………5
3.1.1 Types of endothelium cell …………..……………………………………………………6-8
3.2 VASCULOGENESIS AND ANGIOGENESIS …………………...……………………8-10
4. Vascular Tissue Engineering……………...……………………………………………10-12
4.1 Scaffolds from De-cellularized Matrices ………………………………………………13-14
4.2 Scaffolds from Biodegradable Natural Polymers …………………………...…………15-17
4.3 Scaffolds from Biodegradable Synthetic Polymers ……………………………………17-20
4.4 Body as a bioreactor” approach ………………………………….………………………21
5 Vascularization Strategies for Scaffold ………………..………………………………21-22
5.1 Scaffold Functionalization ………………………………………………..……………22-25
5.2 Cell-Based Techniques …………………………………………….………………………26
5.3 Growth factor-producing cells ………………………………………………………………27
5.4 Bioreactor Design …………………………………………………………………………28
5.5 MEMS-Related Approaches ………………………...…………………………………28-29
5.6 Modular Assembly ………………………………….….………………………………30-31
5.6.1 Vessel-embedded hydrogels ………………………………………………………………31
5.7 In Vivo Systems ……………………………………………………………………………32
5.7.1 Poly-surgery techniques …………………………………………….……………………32
5.7.2 AV loops …………………………………………..…………………………………32-33
Page 3
VIJAYRAJ YANAMALA 1
1. TISSUE ENGINEERING
Tissue Engineering is the study of the growth of new connective tissues, or organs,
from cells and a collagenous scaffold to produce a fully functional organ for implantation
back into the donor host. It also refers to the application of engineering principles to the
design of tissue replacements, usually formed from cells and biomolecules.
Tissue engineering is a fast growing area of research that aims to create tissue
equivalents of blood vessels, heart muscle, nerves, cartilage, bone, and other organs for
replacement of tissue either damaged through disease or trauma. As an interdisciplinary
field, principles from biological, chemical, electrical, materials science, and mechanical
engineering are employed in research and development. Concepts and discoveries from the
fields of molecular and cell biology, physiology and immunology are also readily
incorporated into research activities for tissue engineering. Recent advancements in stem
cell research provide exciting opportunities of using stem cells for regeneration of tissues
and organs.
1.1 PROCEDURE FOR TISSUE ENGINEERED PRODUCT
Typically, an engineered tissue is formed by harvesting a small sample of the
patient’s cells, expanding them in culture, then seeding the cells onto a scaffold
material.
Scaffold materials are intended to define the size and shape of the new “tissue” and
to provide mechanical support for the cells as they synthesize the new tissue.
Scaffolds are usually biodegradable synthetic polymers.
The cell-seeded scaffolds can either be implanted into the patient, with tissue
formation occurring in situ, or cultured further in vitro to achieve properties more
similar to normal tissue before implantation.
This culture period is often carried out in a bioreactor to provide appropriate
mechanical conditioning during tissue formation.
Page 4
VIJAYRAJ YANAMALA 2
2. STRUCTURE OF BLOOD VESSELS
Before tissue engineering a product, it is very important to know the structure,
cellular content, and ECM it is made of, and the signaling molecules it is modelled by.
The wall of an artery consists of three layers.
Intima
Media
Adventitia
Fig1: Artery wall
Reference: http://training.seer.cancer.gov/anatomy/cardiovascular/blood/classification.html
Fig2: Histology of femoral artery
Reference: http://schoolworkhelper.net/histology-labelled-slides/femoral-artery-slide-labelled-
histology/
Page 5
VIJAYRAJ YANAMALA 3
2.1 TUNICA INTIMA
The innermost layer, the tunica intima is simple squamous epithelium surrounded
by a connective tissue basement membrane with elastic fibers.
Endothelial cell monolayer, which prevents platelet aggregation and regulates
vessel permeability, vascular smooth muscle cell behavior, and homeostasis.
A sub-endothelial layer, consisting of delicate connective tissue with branched cells
lying in the interspaces of the tissue
in arteries of less than 2 mm in diameter the sub-endothelial layer consists of a
single stratum of stellate cells, and the connective tissue is only largely developed
in vessels of a considerable size.
An elastic or fenestrated layer, which consists of a membrane containing a net-
work of elastic fibers. This membrane forms the chief thickness of the inner coat.
Fig3: Tunica intima
2.2 TUNICA MEDIA
The middle layer, the tunica media, is primarily smooth muscle and is usually the
thickest layer. It not only provides support for the vessel but also changes vessel
diameter to regulate blood flow and blood pressure.
middle layer is distinguished from the inner layer by its color and by the transverse
arrangement of its fibers.
In the smaller arteries it consists principally of plain muscle fibers in fine bundles,
arranged in lamelle and disposed circularly around the vessel.
It is the thickest layer of all the three layers and contributes to majority of
mechanical strength.
Fig4: Tunica media
Page 6
VIJAYRAJ YANAMALA 4
2.3 TUNICA ADVENTITIA
The outermost layer, which attaches the vessel to the surrounding tissue, is the
tunica externa or tunica adventitia.
This layer is connective tissue with varying amounts of elastic and collagenous
fibers.
The connective tissue in this layer is quite dense where it is adjacent to the tunic
media, but it changes to loose connective tissue near the periphery of the vessel.
The collagen serves to anchor the blood vessel to nearby organs, giving it stability.
Fig4: Tunica adventita
References of Fig2-4: http://www.britannica.com/science/tunica-intima
3. BLOOD VESSEL FORMATION
Vasculogenesis: De novo blood vessel generation from vascular progenitor cells.
Angiogenesis: Formation of new blood vessels via extension or remodeling from existing
capillaries.
Fig5: Circulatory system
Page 7
VIJAYRAJ YANAMALA 5
Fig5: Structural composition of blood vessels
References: Jain R, Nature Med. June 2003
3.1 ENDOTHELIAL CELLS
Almost all tissues depend on a blood supply, and the blood supply depends on
endothelial cells, which form the linings of the blood vessels. Endothelial cells have a
remarkable capacity to adjust their number and arrangement to suit local requirements.
They create an adaptable life-support system, extending by cell migration into almost
every region of the body. Endothelial cells extend and remodel the network of blood
vessels, and help in tissue growth and repair.
Fig6: Endothelial cell culture
References: www.cellapplications.com
Page 8
VIJAYRAJ YANAMALA 6
3.1.1 Types of endothelium cells
i) Human umbilical vein endothelial cell line (HUVEC)
Macrovascular cells
Produce very small amount of VEGF
Dependent on growth factors
limited life span
ii) Human microvascular endothelial cells (HMEC-1)
Main players in angiogenesis
Immortalized cell line
Generate detectable amount of VEGF
Extended life span
New vessels in the adult originate as capillaries, which sprout from existing small
vessels. This process of angiogenesis occurs in response to specific signals. Tissue
vascularizes through an invasion of endothelial cells. Observations such as these reveal
that endothelial cells that are to form a new capillary grow out from the side of an existing
capillary by extending long pseudopodia, pioneering the formation of a capillary sprout
that hollows out to form a tube. This process continues until the sprout encounters
another capillary, with which it connects, allowing blood to circulate.
Fig6: Angiogenesis
References: Molecular Biology of the Cell/ Blood Vessels and Endothelial Cells
Page 9
VIJAYRAJ YANAMALA 7
Endothelial cells have markers that are used to identify the microvasculature in
tissues. Depending of signal that is elicited from ligand attached to receptor on
endothelial cell, vasculogeneis or angiogenesis happen. And the morphology of
endothelial cells too affects the vasculogeneis and angiogenesis. Before experiment is
planned for, it’s important to know the endothelial morphology and the respective
markers it is associated with.
Fig7: Endothelial cells and its markers
Fig8: Morphological differentiation of endothelium cells
Reference: Cleaver O & Melton DA, Nature Med., June 2003
Page 10
VIJAYRAJ YANAMALA 8
Fig9: Morphology of capillaries continuous, fenestrated and discontinuous
Fig10: Morphology of continuous capillary transport and fenestrated capillary transport
3.2 VASCULOGENESIS AND ANGIOGENESIS
Vasculogenesis:
During embryonic development
During adulthood associated with circulating progenitor cells.
Angiogenesis:
Embryonic development
Adulthood: wound healing, menstrual cycle, tumor-angiogenesis.
Fig11: Physiological angiogenesis in adults is restricted
Page 11
VIJAYRAJ YANAMALA 9
It is intriguing to ask why is angiogenesis restricted in adults. The answer is simple, due
to lack of, or reduction of associated growth factors and cytokines.
Fig12: Vasculogenesis, angiogenesis and arteriogenesis
Angioblasts on subjected to bFGF and VEGF are activated and proliferated to
capillaries. This proliferation of angioblast to capillaries can be termed as vasculogenesis.
Vasculogenesis is usually of three phases;
(i) Initiated from the generation of hemangioblasts,
(ii) Angioblasts proliferate and differentiate into endothelial cells.
(iii) Endothelial cells form primary capillary plexus.
When the capillaries are subjected to VEGF and Ang-2, they are activated and
proliferates to blood vessels and its termed as angiogenesis. And these premature blood
vessels are converted by mature blood vessels by the activity of Ang1, bFGF, MCP-1,
PDGF, which is termed as Arteriogenesis.
Blood vessel formation Growth factors
Vasculogenesis Basic Fibroblast Growth Factor, Vascular endothelial growth factor
Angiogenesis Vascular endothelial growth factor, Angiopoietin
Arteriogenesis
Angiopoietin, Basic Fibroblast Growth Factor, Monocyte chemoattractant protein-1, Platelet-derived growth factor
Page 12
VIJAYRAJ YANAMALA 10
Hemangioblast is a multipotent cell, common precursor to hematopoietic and
endothelial cells. Hemangioblast was first hypothesized in 1900. It can be extracted from
embryonic cultures and manipulated by cytokines to differentiate along either
hematopoietic or endothelial route.
Fig13: Hemangioblast proliferates to hematopoietic cells and endothelial cells
Fig14: Formation of vascular network
Page 13
VIJAYRAJ YANAMALA 11
4. Vascular Tissue Engineering
Strategy for TEVG: Basic strategy for vascular tissue engineering consists of the design
and the production of appropriate scaffolds for
Vascular cell adhesion
Proliferation
Differentiation
Choice of cell type
Synthetic materials, for example, polyethylene terephthalate (PET) and expanded poly-
tetrafluoroethylene (ePTFE), are successfully used for the replacement of medium-large
diameter blood vessels (D >6 mm), when high blood flow and low resistance conditions
prevail. The use of PET or ePTFE for small diameter blood vessels leads to several
complications like aneurysm, intimal hyperplasia, calcification, thrombosis, infection, and
lack of growth potential for pediatric applications. These drawbacks are mainly correlated
to the regeneration of a nonfunctional endothelium and a mismatch between the
mechanical properties of grafts and native blood vessels.
Causes of graft failure may be classified into early, midterm, and late.
Early failures: (within 30 days after the implantation) are related to technical
complications, flow disturbances, or acute thrombosis.
Midterm failures: (3 months to 2 years after the implantation) consist of lumen
occlusion due to intimal hyperplasia,
late failures: (>2 years) are related to atherosclerotic disease.
Vascular tissue engineering has become a promising approach to overcome the limits
of autografts morbidity and scarce availability and synthetic grafts inappropriate
properties. Among all requirements for an ideal TEVG, the strictest requisites are
correlated to the regeneration of a functional endothelium and the similarity between
the mechanical proprieties of TEVG and natural blood vessels.
Page 14
VIJAYRAJ YANAMALA 12
Fig14: Vascular tissue engineering
Tissue-engineered vascular graft (TEVG) should mimic the nature’s blood vessels in terms
of bio-compatibility, mechanical properties and processability.
Bio-compatibility Non-toxicity
Non-immunogenicity
Non-thrombogenicity
Non-susceptibility to infection
Ability to grow for pediatric patients
Maintenance of functional endothelium
Mechanical properties Compliance similar to native vessel
Burst pressure similar to native vessel
Kink and compression resistance
Good suture resistance Processability Low manufacturing cost
Readily available with a large variety of length and diameter
Sterilization
Easy storage
Page 15
VIJAYRAJ YANAMALA 13
4.1 Scaffolds from De-cellularized Matrices
De-cellularization process aims to remove all cellular and nuclear matter minimizing
any adverse effects on the composition, biological activity, and mechanical integrity of
the remaining extracellular matrix (ECM) for the development of a new tissue. The
process usually consists of mechanical shaking, chemical surfactant treatment, and
enzymatic digestion. De-cellularized matrix advantages are correlated to its natural three-
dimensional ultrastructure and its structural and functional proteins, essential for cell
adhesion, migration, proliferation, and differentiation.
De-cellularization procedures may remove desirable ECM components, such as collagen,
thus decreasing mechanical properties. Hydrated ECM matrices demonstrate excellent
biomechanical characteristics and improved cellular ingrowth rates.
Fig15: Vascular grafts and its compliance
Fig16: Studies on de-cellularized matrices for vascular tissue engineering
Page 16
VIJAYRAJ YANAMALA 14
Fig17: Studies on de-cellularized matrices for vascular tissue engineering
Fig18: Studies on de-cellularized matrices for vascular tissue engineering
References of Fig14-18: Vascular Tissue Engineering: Recent Advances in Small Diameter Blood
Vessel Regeneration; Valentina Catto, Silvia Farè, Giuliano Freddiand, Maria Cristina Tanzi; ISRN Vascular
Medicine; Volume 2014, Article ID 923030
Page 17
VIJAYRAJ YANAMALA 15
4.2 Scaffolds from De-cellularized Matrices
Natural polymers generally show excellent biological performances; specifically, they
do not activate chronic inflammation or toxicity.
FIBRIN
Fibrin is an insoluble body protein entailed in wound healing and tissue repair.
Fibrin clot, obtained by fibrinogen polymerization due to thrombin, is a fibrillary
network gel that provides a structural support for adhesion, proliferation, and
migration of cells involved in the healing.
Fibrin clot is resorbed through the fibrinolysis, a fibrinolytic process that breaks
down fibrin fibrils.
Fibrinogen may be purified from autologous blood and used for scaffold
fabrication avoiding immunological problems.
ELASTIN
Elastin is one of the major ECM proteins in the arterial wall that confers elastic
recoil, resilience, and durability.
It is an important autocrine regulator to SMC and EC activity, inhibiting migration
and proliferation of SMCs and enhancing attachment and proliferation of ECs.
Elastin, as a coating of vascular devices demonstrated low thrombogenicity with
reduced platelet adhesion and activation.
Elastin co-polymer is made of ePTFE, PET, a copolymer of ePTFE and
polyethylene, and a polycarbonate polyurethane.
HYLAURONAN
Hyaluronan is an anionic non-sulfated glycosaminoglycan (GAG) that consists of
glucuronic acid and N-acetyl Glucosamine
Hyaluronic acid is hydrophilic, non-adhesive, biocompatible, and biodegradable.
SILK FIBRION
Silk fibroin is a protein produced by silkworms and spiders.
The amino acid structure of silk fibroin from Bombyx mori is composed mainly of
glycine (43%), alanine (30%), and serine (12%).
It shows excellent mechanical Properties and biocompatibility.
Silk degrades slowly.
Page 18
VIJAYRAJ YANAMALA 16
COLLAGEN
Collagen is the major ECM protein in the body that supplies mechanical support to
many tissues.
Collagen demonstrates low antigenicity, low inflammatory response,
biocompatibility, biodegradability, and excellent biological properties.
Collagen type I is one of the main components of the vascular wall, whereas it is
widely used as scaffold for vascular tissue engineering applications.
Fig19: Studies on TEVGs fabricated with natural polymers
Fig20: Studies on TEVGs fabricated with natural polymers
Page 19
VIJAYRAJ YANAMALA 17
Fig21: Studies on TEVGs fabricated with natural polymers
4.3 Scaffolds from Biodegradable Synthetic Polymers.
Biodegradable synthetic polymers generally demonstrate tailorable mechanical
properties and high reproducibility, compared to natural polymers, can be produced in
large amounts.
POLY-GLYCOLIC ACID
PGA is a semi-crystalline, thermoplastic aliphatic polyester synthesized by the ring-
opening polymerization of glycolide.
It degrades rapidly in vivo by hydrolysis to glycolic acid, metabolized and
eliminated as carbon dioxide and water, and completely degrades in vivo within 6
months.
Page 20
VIJAYRAJ YANAMALA 18
POLY-LACTIC ACID
PLA is a thermoplastic aliphatic polyester that demonstrates good biocompatibility
and mechanical properties and the ability to be dissolved in common solvents for
processing
PLA is more hydrophobic than PGA, leading to a slower degradation rate.
PLLA takes months or even years to lose its mechanical integrity
POLY-𝜀-CAPROLACTONE
PCL is a semi-crystalline, aliphatic polyester synthesized by the ring-opening
polymerization of 𝜀-caprolactone.
It shows good mechanical properties, specifically high elongation and strength, and
good biocompatibility.
Furthermore, PCL degrades very slowly in vivo by enzymatic action and by
hydrolysis to caproic acid and its oligomers.
It takes more than 1 year to completely degrade in vivo.
POLY-GLYCEROL SEBACATE
PGS is an elastomer synthesized by poly-condensation of glycerol and sebacic acid.
It demonstrates good biocompatibility and good mechanical properties, specifically
high elongation and low modulus, indicating an elastomeric and tough behavior.
Fig22: Studies on TEVGs fabricated with biodegradable synthetic polymers
Page 21
VIJAYRAJ YANAMALA 19
Fig23: Studies on TEVGs fabricated with biodegradable synthetic polymers
Fig24: Studies on TEVGs fabricated with biodegradable synthetic polymers
Page 22
VIJAYRAJ YANAMALA 20
Fig25: Studies on TEVGs fabricated with biodegradable synthetic polymers
Fig26: Studies on TEVGs fabricated with biodegradable synthetic polymers
Page 23
VIJAYRAJ YANAMALA 21
4.4 Body as a bioreactor” approach
In 2001, Shinoka and coworkers reported the first application of a tissue engineered
blood vessel in a human. Cells were harvested from patient's peripheral vein and cultured
for 10 days on a tubular scaffold made from polycaprolactone–polylactic acid copolymer
that was reinforced with PGA. The engineered blood vessel was subsequently implanted
as a pulmonary artery graft into the patient and remained patent for at least 7 months.
However, compared with other engineered blood vessels, BM-MNC-seeded grafts can
only be used in a low-pressure circulatory system, due to the lack of mature ECM and
mechanical strength prior to implantation.
5 Vascularization Strategies for Scaffold
The biggest challenge in the field of tissue engineering remains mass transfer
limitations. This is the limiting factor in the size of any tissue construct grown in vitro.
Within the body, most cells are found no more than 100–200mm from the nearest
capillary, with this spacing providing sufficient diffusion of oxygen, nutrients, and waste
products to support and maintain viable tissue. Likewise, when tissues grown in the
laboratory are implanted into the body, this diffusion limitation allows only cells within
100–200mm from the nearest capillary to survive.
Thus, it is critical that a tissue be pre-vascularized before implantation with proper
consideration given to the cell and tissue type, oxygen and nutrient diffusion rates, overall
construct size, and integration with host vasculature. In the laboratory, limited diffusion
of oxygen is the primary reason that construction of tissues greater than a few hundred
microns in thickness is currently not practicable.
Approaches to address this problem generally fall into six major categories:
scaffold functionalization,
cell-based techniques,
bioreactor designs,
(d)microelectromechanical systems(MEMS)–related approaches,
modular assembly,
in vivo systems.
Scaffolds may be functionalized through different angiogenic factor loading
techniques or through increased porosity or channeling of scaffolds to form perfusion
elements. Use of MEMS and microfluidic technologies to recapitulate the branching
Page 24
VIJAYRAJ YANAMALA 22
network of the microvasculature is an alternative approach being pursued in many labs,
with systems generally formed from nondegradable materials such as silicone.
Fig 27: Schematic diagrams of different vascularization approaches. (A) Scaffold
functionalization. (B) Cell based technique, (C) Bioreactor design, (D) Micro-electro-
mechanical system, (E) Modular assembly, (F) In-vivo systems.
5.1 Scaffold Functionalization
One of the classical approaches to producing larger tissues has been to decorate or
supplement scaffolds, either natural or synthetic, with pro-angiogenic factors such as
VEGF, basic fibroblast growth factor (bFGF), or PDGF. This mimics the in vivo condition
where these factors are associated with the extracellular matrix (ECM) to stabilize
conformation and protect from proteolytic digestion.
Beyond these basic scaffold-loading approaches, protein modification techniques
have been applied to scaffolds by forming binding domains for angiogenic factors via
Page 25
VIJAYRAJ YANAMALA 23
fusion proteins or coupling using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)
and N-hydroxysuccinimide (NHS) chemistry. Fusion proteins composed of hepatocyte
growth factor and a collagen-binding domain have been used to facilitate loading of
hepatocyte growth factor, subsequently promoting capillary formation in gel culture in
vitro as well as blood vessel growth in vivo. Likewise, fusion proteins of bFGF and fibrin-
binding peptide Kringle1, or PDGF and collagen-binding domains, have been utilized to
couple bFGF or PDGF to fibrin or collagen gels.
Fig 28: Currently used growth/signaling factor
The polymer encapsulated PDGF had evenly distributed throughout the scaffold and
was released more slowly through bulk degradation. Synthetic microsphere
encapsulation has also been used to trap bFGF in PLGA, incorporating these microspheres
into alginate scaffolds or simply injecting them with small intestinal submucosa and pre-
adipocytes, both of which have been shown to significantly enhance vascularization.
Fig29: Scaffold Functionalization Techniques for Vascular Tissue Engineering
In natural vasculogeneis, differentiation and formation of angioblasts into primitive
blood vessels is induced by VEGF receptor activation, with concentration gradients and
maintenance of threshold levels required for differentiation and angiogenesis. Sprouting
of new vessels through angiogenesis is then induced by angiopoietins, ligands for the
endothelial cell receptor kinase TIE, which modulates VEGF activity and may direct
Page 26
VIJAYRAJ YANAMALA 24
angiogenesis through the pattern of signaling by VEGF and TIE receptors. Further
branching and remodeling is controlled by matrix metalloproteinase activity, influencing
cell migration and differentiation through the release of pro-angiogenic factors within the
matrix. Scaffold design should apply this knowledge during vessel development in vivo to
form biomaterial scaffolds loaded with these factors, that has control over release rates
over time and thus vascular development.
One of the way to address the uniform oxygen diffusion is through the use of
channeled scaffolds. Channeled scaffolds have been formed by incorporating phosphate-
based glass fibers into collagen scaffolds. By incorporating phosphate-based glass fibers
into collagen scaffolds, channel size and distribution is controllable based on the original
size of the glass fibers (10–50mm) and the fiber-to-fiber spacing. Thus, when these fibers
are degraded, micro-channels are left behind that offer potential for flow and improved
cell viability.
Besides micro-channeling, directing scaffold vascularization through micro-patterning
or molecular gradients has been explored. Scaffolds were produced by mixing the poly-
caprolactone with PLGA micro nanoparticles and casting the polymer onto a grooved
surface before leaching out the micro nano spheres. This process produces surfaces that
are conducive for vascular cell alignment and also increase medium diffusion.
By stacking these layers, it may be possible to build up three-dimensional (3D) tissues
with cellular organization for blood vessel formation, an outcome that may also be
directed by forming gradients within the scaffold.
Controlled spatial deposition of different biopolymers, growth factors, and cells may
also be achieved as multi-nozzle systems have been developed. Overall, these scaffolding
techniques offer fine control over vascularization potential and offer a multitude of
options in scaffold design and engineering to create functional tissue outcomes.
Future scaffold designs may be improved through the use of an emerging tool,
computer-aided tissue engineering, which can help model and design scaffolds with
controlled internal and external architecture, particularly vascular channel elements of
different sizes and shapes.
Page 27
VIJAYRAJ YANAMALA 25
Fig30: Multi-nozzle deposition for construction of 3D biopolymer tissue scaffolds
Reference: Khalil, J. Nam and W. Sun Laboratory for Computer-Aided Tissue Engineering, Department
of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, Pennsylvania, USA; Multi-
nozzle deposition for construction of 3D biopolymer tissue scaffolds S; Rapid Prototyping Journal Volume
11. Number 1.2005.9–17
Fig 31: Fabrication of channeled scaffolds with ordered array of micro-pores through
microsphere leaching
Reference: J. Y. Tan & C. K. Chua & K. F. Leong; Fabrication of channeled scaffolds with ordered array
of micro-pores through microsphere leaching and indirect Rapid Prototyping technique; Biomed
Microdevices (2013) 15:83–96 DOI 10.1007/s10544-012-9690.
Page 28
VIJAYRAJ YANAMALA 26
5.2 Cell-Based Techniques
To help compensate for issues with growth factor delivery, co-cultures with
endothelial cells have been utilized to provide a starting point for vascularization,
endothelial cells are introduced into the tissues via 3D multicellular spheroids or simple
mixing of cultures. Endothelial cell spheroids produce capillarylike sprouts, especially in
the presence of angiogenic factors such as VEGF and bFGF, or in coculture with
fibroblasts, but sprout diameter and length was reduced in cocultures of endothelial
cells and osteoblasts.
Beyond spheroid cultures, simple cocultures of endothelial cells, fibroblasts, and
other cell types have been used to grow vascularized skin, skeletal muscle, and bone
tissues, among others. In several cases, the role of fibroblasts is critical for the formation
and the maintenance of the microvasculature.
Fig 32: Endothelial cell markers
In one research paper, team had made scaffold vascularized by combining layers of
endothelial cells and layers of other cells, such as fibroblasts, within native hydrogels.
Another team had made spacing a layer of dermal fibroblasts at a distance 1.8–4.5mm
from human umbilical vein endothelial cell–coated beads within a fibrin gel fed with
media containing VEGF and bFGF. Endothelial cells produced capillaries based on the
distance of the endothelial cells from the fibroblasts.
Page 29
VIJAYRAJ YANAMALA 27
5.3 Growth factor-producing cells
An additional cell-based approach that has become a focus of vascular research is the
transfection of cells to overexpress angiogenic factors. These cells can be seeded within
biomaterial scaffolds and release cytokines that modulate vascular cell migration,
proliferation, and maturation into tubular vessels in a more controlled, biomimetic
manner than simple scaffold loading.
In a study aimed at producing tissue engineered bone, combinations of scaffolds were
coated with or without VEGF-plasmid DNA and loaded with hMSCs transfected with or
without the VEGF plasmid. Compared to controls, VEGF plasmid–coated scaffolds and
VEGF-transfected cells demonstrated significantly enhanced vascularization,
osteogenesis, and scaffold resorption compared to control groups, with the VEGF-
transfected cells producing the highest rate of vascularization.
Advantage: As opposed to growth factor scaffold-loading–based techniques, these cell-
based approaches demonstrate significant potential for sustained growth factor release
over time and better overall vascularization.
Fig33: Mouse VEGF-C Gene cDNA Clone
Reference: http://www.sinobiological.com/VEGF-C-cDNA-Clone-g-12019.html
Page 30
VIJAYRAJ YANAMALA 28
5.4 Bioreactor Designs
In generating tissues in vitro, bioreactor systems are often used to perfuse culture
medium through a porous scaffold to try to maintain cell viability in the middle and
homogeneity throughout the construct.
Rotating bio-reactors: In terms of vascularized tissue engineering, early events (growth
and differentiation) of ocular angiogenesis have been studied in these bioreactors, using
human retinal cells and bovine endothelial cells in co-culture on micro-carrier beads and
grown in the horizontally rotating bioreactors for up to 5 weeks. In co-culture, the
endothelial cells formed cords and capillary-like structures as well as the beginning of
sprouts, indicators of a developing vasculature.
Perfusion bioreactors: Vascular perfusion bioreactors typically focus on producing a
tissue-engineered blood vessel, not on vascularizing another tissue type. Using pulsatile
conditions typically found in vivo, functional arteries may be grown in vitro. The perfused
tissues displayed enhanced cell viability and increased metabolic activity when compared
to non-perfused controls.
Fig34: Bio-reactor design for vascular tissue engineering
5.5 MEMS-Related Approaches
Microfabrication techniques have gained popularity as they offer fine control over
the formation of a microvascular network. These capillary networks may be perfused
and endothelialized, providing a mimic of natural vasculature as well as oxygen and
nutrient delivery and waste removal. Standard techniques use plasma etching or
lithographic techniques to produce desired features with micron-scale precision, with
replica molds of PDMS cast from the negative feature molds. These molds may then be
bonded with one another before seeding with endothelial cells to form cylindrical
capillary channels.
Page 31
VIJAYRAJ YANAMALA 29
Fig 35: Fabrication process for collagen-fiber reinforced elastin-mimetic composite tissue
scaffold by MEMS
Reference: Dr. Nisarga Naik, Dr. Jeffrey Caves, Prof. Elliot Chaikof; Generation of Spatially Aligned
Collagen Fiber Networks through Microtransfer Molding; Adv Healthc Mater. 2014 March; 3(3): 367–
374. doi:10.1002/adhm.201300112
Direct-write laser technology has been utilized to form multiple-depth channel
systems with diameter changes between parent and daughter vessels that mimic
physiological systems. Devices are produced using similar techniques as synthetic
microfluidics, with PGS layers formed by casting onto negative molds, released using a
sacrificial layer, and bonded together by physically adhering and curing the films under
vacuum. By subsequently stacking single-layer microfluidic networks, with consideration
for oxygen limitations, 3D scaffolds with complex vascular micro-channels can be
produced for different tissue types.
By casting silk onto negative molds, treating with methanol to form micro molded water
stable films, and binding to a flat layer using aqueous silk solution, microfluidic devices
composed entirely of silk protein are produced. These devices have demonstrated
improved mechanical properties compared to PGS films and also support hepatocyte
culture, making these a promising option for degradable microfluidics.
Fig 36: MEMS approach to vascular tissue engineering
Page 32
VIJAYRAJ YANAMALA 30
5.6 Modular Assembly
An emerging technique for producing pre-vascularized tissues involves the modular
assembly of endothelialized micro-tissues to form a macro-tissue.
Fig37: Bottom-up & Top-down approaches to tissue engineering. In the bottom-up approach
there are multiple methods for creating modular tissues, which are then assembled into
engineered tissues with specific micro architectural features. In the top-down approach, cells
and biomaterial scaffolds are combined and cultured until the cells fill the support structure to
create an engineered tissue.
Fig38: Creation of human blood vessel from cell sheet technology. Fibroblast cells are harvested
from the patient, expanded into cell sheets, wrapped and cultured around a cylindrical mandrel
to create robust, blood vessels (A). After seeding w/harvested endothelial cells, the grafts were
tested as AV shunts for dialysis patients (B, C), where they performed well through multiple
puncture wounds
Reference of Fig 37,38: Jason W. Nichol and Ali Khademhosseini; Modular Tissue Engineering:
Engineering Biological Tissues from the Bottom Up; Soft Matter. 2009 ; 5(7): 1312–1319.
doi:10.1039/b814285h
Page 33
VIJAYRAJ YANAMALA 31
These packed-bed macro tissues can be perfused with medium or whole blood, or be
connected to host vasculature through the use of a chicken chorio allantoic membrane
assay. This technique has been used to generate tissues using hepatocytes, chondrocytes,
and SMCs.
Fig39: Chorioallantoic membrane vascular assay (CAMVA)
However, while these modular tissues demonstrate the ability to be perfused as well as
integrate with host vasculature over time, they do not replicate the tree-like vasculature
exhibited in vivo and do not facilitate tissue integration in vivo by providing components
that enable immediate anastomosis to host vasculature.
5.6.1 Vessel-embedded hydrogels
Another developing modular approach is the perfusion of single- or multi-channel
hydrogels. These systems are promising given that they can address two of the underlying
issues with vascularization, measurement of oxygen nutrient diffusion, and connection
with host vasculature. These micro vessels were produced by embedding a 120-mm-
diameter needle within a collagen gel, removing it, and seeding with endothelial cells.
Fig40: Modular Assembly Approaches to Vascular Tissue Engineering
Reference of Fig 14-29,32,34,36,37,40: Michael Lovett, Ph.D.,1 Kyongbum Lee, Ph.D,Aurelie
Edwards, Ph.D, and David L. Kaplan, Ph.D, Vascularization Strategies for Tissue Engineering; TISSUE
ENGINEERING: Part B Volume 15, Number 3, 2009 ª Mary Ann Liebert, Inc. DOI:
10.1089=ten.teb.2009.0085
Page 34
VIJAYRAJ YANAMALA 32
5.7 In Vivo Systems
5.7.1 Poly-surgery techniques
Beyond efforts to build vascularized tissues in vitro, researchers have used cell sheet
engineering and poly-surgery techniques to produce tissues up to 1mm in thickness (Table
8). Cell sheet engineering techniques have been used in corneal surface reconstruction,
blood vessel grafts, and myocardial tissue engineering, among others. To form
vascularized tissue, confluent sheets of tissue cells can be grown and stacked to form
tissue. To overcome limitation of vascularization of thick tissues, the layered cell sheets
were transplanted into rats and allowed to vascularize over a period of 1–3 days. Upon
complete vascularization of the transplant, another cell sheet was added and
vascularized, continuing in this layer-by-layer transplantation approach until required
thickness is achieved.
5.7.2 AV loops
In this intrinsic vascularization model, a vein or synthetic graft is used to form a shunt
loop between an artery and a vein and is enclosed within a chamber that is either empty
or housing an ECM scaffold to be vascularized.
Fig41: A typical shunt loop
Reference: www.gefaesszentrum-bremen.de
Page 35
VIJAYRAJ YANAMALA 33
In an experiment empty AV loop was used in a rat model, where constructs formed
extensive arteriole–capillary– venule networks within a fibrin matrix exuded from the AV
loop, with initial development occurring between 7 and 10 days and maturing over time.