The document discusses vascular tissue engineering and strategies for scaffold vascularization. It begins by covering tissue engineering procedures and the structure of blood vessels. It then discusses blood vessel formation through vasculogenesis and angiogenesis. Various vascular tissue engineering approaches are examined, including using decellularized matrices, natural polymers like fibrin and collagen, and biodegradable synthetic polymers. Strategies to vascularize scaffolds are outlined, such as scaffold functionalization with growth factors, incorporating endothelial cells, designing channeled scaffolds, and using growth factor-producing cells.

1. DIFFUSION BASED AND
VASCULAR CONSTRUCTS,
TRANSPORT OF NUTRIENTS
AND METABOLITES
YANAMALA VIJAY RAJ
BT14M004
MTECH IN CLINICAL ENG
IIT Madras & CMC Vellore & SCTIMST

2. CONTENT
1 TISSUE ENG
2 STRUCTURE OF BLOOD VESSEL
3 BLOOD VESSEL FORMATION
4 VASCULAR TISSUE ENGINNERGING
5 VASCULARIZATION STRATEGIES FOR SCAFFOLD

3. 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.

4. 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.

5. 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

6. 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.
 An elastic or fenestrated layer, which consists of a membrane containing a network
of elastic fibers. This membrane forms the chief thickness of the inner coat.

7. 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.
 It is the thickest layer of all the three layers and contributes to majority of
mechanical strength.

8. 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.

9. 3. BLOOD VESSEL FORMATION
 Vasculogeneis: De novo blood vessel generation from vascular progenitor cells.
 Angiogenesis: Formation of new blood vessels via extension or remodeling from
existing capillaries.

10. 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.

11. 3.1 ENDOTHELIAL CELLS
 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

12. 3.1 ENDOTHELIAL CELLS
 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.

13. 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.
Physiological angiogenesis in adults is restricted

14. 3.2 VASCULOGENESIS AND ANGIOGENESIS
 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.
Vasculogenesis, angiogenesis and arteriogenesis

15. 3.3 Formation of vascular network

16. 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

17.  Synthetic materials, for example, polyethylene terephthalate (PET) and expanded
polytetrafluoroethylene (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.

18. Vascular tissue engineering
Tissue-engineered vascular graft (TEVG) should mimic the nature’s blood vessels in terms of
bio-compatibility, mechanical properties and processability

19. 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
Studies on de-cellularized matrices for vascular tissue engineering

20. Scaffolds from Natural Polymers
 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.
 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

21.  SILK FIBRION
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.

23. Scaffolds from Biodegradable Synthetic Polymer
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
ringopening 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

24. POLY-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.
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.

26. 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.

27. 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

28. 5 Vascularization Strategies for Scaffold
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.

29. 5 Vascularization Strategies for Scaffold
Approaches to address this problem generally fall into six major categories:
5.1 Scaffold functionalization,
5.2 Cell-based techniques,
5.3 Bioreactor designs,
5.4 Microelectromechanical systems(MEMS)–related approaches,
5.5 Modular assembly,
5.6 In-vivo systems.

30. 5.1 Scaffold Functionalization

31. 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.
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 preadipocytes, both of which have been shown
to significantly enhance vascularization.

32. 5.1 Scaffold Functionalization

33. 5.1 Scaffold Functionalization
VEGF receptor -> natural vasculogeneis, differentiation and formation of angioblasts
into primitive blood vessels
Angiopoietins -> Sprouting of new vessels through angiogenesis
VEGF and TIE receptors: direct angiogenesis
Release of pro-angiogenic factors -> cell migration and differentiation
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.

34. 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.

35. Channeled scaffolds
Cristina Martı Et all 2012
Channeled scaffolds implanted in adult rat brain

36. Cell-Based Techniques

37. 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 capillary like 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

38. Cell-Based Techniques
 A research 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.

39. 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.
 VEGF plasmid–coated scaffolds and VEGF-transfected cells demonstrated significantly
enhanced vascularization, osteogenesis, and scaffold resorption
 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.
Mouse VEGF-C Gene cDNA Clone

40. 5.5 Microfabrication techniques
 Microfabrication techniques have gained popularity as they offer fine control over
the formation of a microvascular network.
 These capillary networks may be perfused and endothelialzed, providing a mimic of
natural vasculature as well as oxygen and nutrient delivery and waste removal.
 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.

41. 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

42. Modular Assembly
 An emerging technique for producing pre-vascularized tissues involves the modular
assembly of endothelialized micro-tissues to form a macro-tissue

43. 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
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.

44. 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.
 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.

45. A. D. Bach, A. Arkudas, J. Tjiawi, E. Polykandriotis, U. Kneser, R. E. Horch, J. P. Beier *; A new approach to tissue engineering of vascularized skeletal muscle;
J. Cell. Mol. Med. Vol 10, No 3, 2006 pp. 716-726