DIFFUSION BASED AND VASCULAR CONSTRUCTS, TRANSPORT OF NUTRIENTS AND METABOLITES

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

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

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.

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/

http://training.seer.cancer.gov/anatomy/cardiovascular/blood/classification.html

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

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

http://www.britannica.com/science/tunica-intima

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

http://www.cellapplications.com/

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

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

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

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

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


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.


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


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




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


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.


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




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.


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








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


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


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


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.


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.


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.


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

http://www.sinobiological.com/VEGF-C-cDNA-Clone-g-12019.html


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.


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


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


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


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

http://www.gefaesszentrum-bremen.de/dialyse/shunts/kunststoff-shunts/


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.

Health & Medicine

DIFFUSION BASED AND VASCULAR CONSTRUCTS, TRANSPORT OF NUTRIENTS AND METABOLITES