Material-based Cell Delivery Systems

Contents

1 Introduction....................................................................................................................3

1.1 Regenerative Medicine and scaffolds function........................................................3

1.2. Endothelial Progenitor Cells...................................................................................4

1.2.1.Endothelial Progenitor Cells for vasculogenesis...............................................6

1.3 Biomaterials for neovascularization........................................................................7

2. Material-based deployment enhances efficacy of endothelial progenitor cells.............9

2.1 Characterization of EPC and OEC...........................................................................9

2.2 Scaffold..................................................................................................................10

2.3 Cotransplantation of EPC and OEC Enhances Neovascularization in Vivo.........11

3. Discussion....................................................................................................................13

4. Other applications of tissue engineering and future visions........................................14

5. Bibliography................................................................................................................16

Regenerative Medicine

Without Scaffolds

With Scaffolds

Cell Therapy

Tissue Engineering

Fig. 1 – Classification of regenerative medicine based on the use of scaffold.

1 Introduction

Probably the first reference to “regeneration” is the story of the Greek Titan,

Prometheus. Punished by Zeus, Prometheus was ordered to be chained to a rock, and

that an eagle would eat his liver each day. However, Prometheus liver was able to

regenerate itself daily, enabling him to survive. The main goal of cells and tissue

engineering today and for the future, is I think the development of novel therapies to

restore lost, damaged or aging cells and tissues in the human body equalizing the

primordial concept of “regeneration”.

Tissue engineering is a new filed that is rising in importance in the biomedical

engineering. It is an interdisciplinary area between cellular and molecular biology and

materials, chemistry and mechanical engineering. The possibility of manipulating and

reconstruct a tissue function has tremendous applications in clinical and drug testing

studies. Tissue engineering, is likely to play a key role in cell and gene therapies during

the next few years in addition to expanding the possibilities of needed transplantations.

(Bernhard Palsson, 2003)

1.1 Regenerative Medicine and scaffolds function

When a tissue or organ is damaged, not only the cellular functions are affected

but also the extracellular matrix (ECM) is affected. A tissue has a given function, and in

order to work properly, the interplay between cells and the ECM must be dominant. So,

in order to create a neotissue, cells in culture most be provided with an artificial or

biological ECM. Cells can only specialize into a limited degree when placed as a

suspension on tissue, because they need a template that guides their expansion,

specialization and organization. In tissue engineering the substitute of the ECM is called

a “scaffold”. The scaffold provides a three dimensional support, in order to cells

infiltrate and

proliferate into the

targeted functional

tissue or organ.

Cells that are

Fig.2 - Cells can be isolated from the patient’s body, and expanded in a petridish in laboratory. Once we have enough number of cells, they can be seeded on a polymeric scaffold material, and cultured in vitro in a bioreactor or incubator. When the construct is matured enough, then it can be implanted in the area of defect in patient’s body.

incorporated in the scaffold are then implanted in the body, and hopefully they will

grow to specific functions (Ikada, 2006). Besides using scaffolds, cell-based therapies

are commonly used with only cells infusions that can still support the safety of the cells,

but have a high rejection rate by the patient, and will rapidly die once inside the body

(Fig.1) (Hofmann M, 2005).

A biomaterial can be designed to mediate the differentiation and proliferative

capacity of the cells in the implant. Some proteins can be adsorbed to the material

surface. Using embryonic stem (ES) cells, it is possible to control the differentiation of

these cells since intracellularly there are receptor-ligand proteins that will interact with

the proteins adsorbed. This will lead to cytoskeletal modulation and finally to gene

modulation, proliferation and synthesis (Wen Jie Zhang, 2007).

For this particular work, it is

important to study how material-based

deployment can possible enhance

efficacy endothelial progenitor cells

(EPC) for revascularizing of a vascular

network. So first, we are going to

study what are endothelial progenitor

cells, then what kind of absorbable

biomaterials that exist and can be used, and finally we will analyze a set of experimental

results, so we can understand the application of tissue engineering (Fig.2) in

revascularization.

1.2. Endothelial Progenitor Cells

Stem cells are found in most, if not all, multi-cellular organisms. The capacity to

differentiate into specialized cell types, defines the potency of the stem cells. So this

requires stem cells to be either totipotent, pluripotent, that can differentiate into cells

from all the embryonic germ layers and multipotent that can differentiate into only a

limited range of cell types (Scholer, 2007) .Other cells, named as progenitor cells, can

divide a limited number of times before facing a change in their potency or undergoing

differentiation (Ahmed, 2009). Stem cells can be grown and transformed into

specialized cells with different characteristics according to their lineage. There are

different sources for plastic adult stem cells, such as umbilical cord blood and bone

marrow that can be used in medical therapies

EPC are blood circulating cells derived from bone marrow that have the ability

to differentiate into endothelial cells, these are responsible for the formation of blood

vessels.  Endothelial progenitor cells found in adults are thus related to angioblasts,

which are the stem cells that form blood vessels during embryogenesis. This process of

forming blood vessels is known for vasculogenesis. (Asahara T e. a., 1997). Endothelial

progenitor cells are thought to participate in pathologic angiogenesis such as that found

in retinopathy and tumor growth. Angiogenesis and vasculogenesis are very similar but

are different in one aspect: The term angiogenesis refers to the formation of new blood

vessels from pre-existing ones, so if a monolayer of endothelial cells begin to proliferate

to form capillaries, angiogenesis is occurring. As endothelial progenitor cells are

originally derived from the bone marrow, it is thought that various cytokines, growth

factors, and hormones cause them to be mobilized from the bone marrow and into the

peripheral circulation where they ultimately are recruited to regions of angiogenesis

(Fig. 3) (Asahara T e. a., 1999).

1.2.1.Endothelial Progenitor Cells for vasculogenesis

Cardiovascular pathologies, such as myocardial infarction and ischemic

diseases, are the number one cause of death globally. (Krenning, 2009.). After an

Fig. 3 - Endothelial progenitor cells (EPCs) circulate in adult human peripheral blood and are mobilized from bone marrow by cytokines, growth factors, and ischemic conditions. Vascular injury is repaired by both angiogenesis and vasculogenesis mechanisms. Circulating EPCs contribute to repair of injured blood vessels mainly via a vasculogenesis mechanism. [Adapted from Murasawa, 2004]

Fig.4 - Mechanisms of angiogenesis and vasculogenesis. (a) Sprouting angiogenesis originates from the pre-existing vasculature (i) and encompasses the secretion of matrix metalloproteases that breakdown the vascular basement membrane (ii) and allow migration of endothelial cells (ECs, yellow) (iii). Proliferation and subsequent migration of newly-formed ECs results in the formation of a solid endothelial cord (iv). Lumen formation and stabilization are the final processes of sprouting angiogenesis (v). (b) Vasculogenesis begins with the formation of a primary vascular plexus by endothelial progenitor cells (EPCs) (i,ii). Matrix deposition (iii) and lumen formation (iv) by EPCs result in the formation of immature capillaries.

ischemic damage, it is mounted a local inflammatory response that is important for the

clearance of cell debris and to preserve the tissue integrity. (van Amerongen, 2007) So

the preservation of local vasculature, or even induction of vascularization might

decrease the lesion size, prevent cell apoptosis, and might inhibit organ failure. Though,

EPC are being used in clinical trials today, for the induction of neovascularization.

Adult neovascularization, is normally regarded to

be a consequence of angiogenesis, rather than

vasculogenesis. Along with angiogenesis, it occurs an

increase in vasopermeability, leading to the

extravasation of plasma proteins that function as a

temporary scaffold for migrating endothelial cells

(EC). The endothelium segregates some matrix

metalloproteases, that break the vascular basement

membrane allowing EC to migrate (Fig. 4.a) (Risau,

1997). In the other hand, Physiological EPC-induced

neovascularization is initiated by hypoxia (Fig. 4.b).

Resuming, EPC-driven neovascularization starts with

the mobilization of EPC from the bone marrow, to the circulating blood in response to a

stress pr damage-related signals (e.g. vascular endothelial growth factor [VEGF],

stromal cell-derived factor 1 [SDF-1] and monocyte chemotatic protein [MCP-1]. Then

migration of EPC occurs through the bloodstream and finally extravasation of EPC

Table 1. Clinical trials and animal models for cardiovascular

through the endothelium. New vessels are then formed, once the cells migrate to the site

of neovascularization (Jujo, 2008).

1.3 Biomaterials for neovascularization

As described above, bioartificial implants containing cells have a large number

of clinical applications in treatment of diseases (Unger RE, 2005). During the past five

years the potential of EPC for the treatment of ischemic diseases has been studied, in

which a patient’s own cells have been isolated, and reinfused. Typically, more than 90%

of the transplanted cells in this manner will rapidly die, and there is no control of the

cells once they are inside the body (Hofmann M, 2005.). Besides these studies

supporting the safety of these cells, they indicate that simple infusions may have som

limitations in the treatment success. An alternative is the use of sophisticated material

carriers that promote tissue formation by cells by using the material as template. The

success of biomaterials, requires a balance between providing proper immune

protection, and minimizing mass transport resistances for oxygen and nutrients. Growth

supplements such as endothelial cell growth factor (ECGF) and vascular endothelial

growth factor (VEGF165) have been used to stimulate endothelial cell migration,

proliferation and survival (Lee KY, 2003). In order to obtain a successful

vascularization it is required the appropriate delivery of substances. Alginate gels have

proven to be appropriate materials in controlling the VEGF release, and also are

biocompatible once inside the body (Peters MC, 1998).

Alginate is a naturally occurring polysaccharide, being formed by α-L-guluronic

and β-D-mannuronic acid residues.

For the current work it was used to fabricate the scaffolds as it had been used as

a delivery vehicle in the past. Besides this, and in order to have control over cell

adhesion, proliferation and cell fate after transplantation, it was covalently coupled

peptides containing arginine-glycineaspartic acid (RGD) amino sequence to the

alginate. The polymer used is normally non-adhesive, RGD is an ubiquitous cell-

binding domain found in many extracellular matrix molecules, conferring a specific

mechanism for integrin-mediated cell adhesion (Shin H, 2003). It was also used several

forms of VEGF as a component of the material system since it is important in the

Fig.5 - Origin and fate of endothelial progenitor cells (EPCs). Schematic overview of the proposed lineage, cell surface markers and differentiation of EPCs. CD14+ EPCs originate from the myeloid cell lineage and coexpress marker proteins from the myeloid cell and endothelial cell (EC) lineages. CD34+ progenitor cells originate directly from hematopoietic stem cells and exclusively express markers from the EC lineage.

vascular growth and formation. VEGF121 and VEGF165 were the two isoforms most

obtainedand the differ in the presence of an heparan sulfate binding site, with the result

that VEGF121 is a very high defusable protein in contrast to VEGF165, which bonds

moderately to extracellular matrix (Carmeliet, 2000)

Nowadays it have been studied a variety of cells such as, cardiac stem cells,

natural killer cells, bone marrow cells, dendritic cells, and EPC, in clinical

revascularization trials. EPC are isolated and purified according to the expression of

CD34 and VEGF-2, extracellular markers, that are found in hematopoietic cells

population contributing for revascularization of damaged tissue (Ingram DA, 2004).

Another kind of cells can be isolated from mononuclear cells, this cells are named

outgrowth endothelial cells (OEC) and can also have applications in this area. The

interesting of these cells is also that they maintain a high proliferative potential and also

present some endothelial

cell markers, including

CD31 and VEGFR-2

(Fig.5) (Ingram DA,

2004). For this work, it

was studied the capacity of

the releasing biomaterial

system for supporting

these two types of cells

(EPC and OEC), in order

to reverse severe hindlimb

ischemia.

2. Material-based deployment enhances efficacy of endothelial

progenitor cells

2.1 Characterization of EPC and OEC

For this work it was studied both EPC and OEC for their potential utility in

relieving ischemia, contributing to angiogenesis. These two types of cells differ in their

morphology, EPC are round shaped cells that form cord-like structures, OEC have

cobblestone-like morphology, similarly to human microvascular endothelial cells and

expressing high telomerase activity (Fig.6 ).

FACS analysis, confirmed that EPC were monocyte/macrophage lineage cells and OEC

were vascular endothelial lineage cells. OEC expressed endothelial cell surface antigens

including CD31, CD144 but were negative for CD34. EPC also expressed CD31,

CD144, VEGFR-2. CD14, which is a monocyte (macrophage cell surface antigen, was

only expressed in EPC. The role of both kinds of cells was studied by the in vitro cell

sprouting assay. Cells were carried in a bead, and then it was studied the formation of

capillary-like extensions. As shown in the (Fig.7), coculture of EPC, OEC and EC

resulted in high sprouting of cells, and contrasted with the absence of sprouts when EPC

were cultured alone. It was also studied the protein expression values, and both in OEC

and EPC it was observed high levels of angiogenic factors, FGF-, IL-12 and IP-10.

Fig.6. On the left, Telomerase activity of cultured EPCs, OECs, and ECs (passage 6). Values represent mean and standard

deviation. On the right, Photomicrographs of cultured EPCs and OECs isolated from human umbilical cord blood.

Fig.7 - The cell types were cultured alone or in various combinations to examine their ability to participate or effect sprout . ECs alone form sprouts (arrows) when immobilized on microcarrier beads that

). Culture of EPCs alone led to no sprouting (Upper Center), and OECs alone exhibited ). Combining ECs and OECs significantly increased sprout formation, and

increased lumen formation (areas delineated by yellow dashed lines) in the sprouts (Lower Left). Coculture of EPCs, OECs, and ECs led to significant sprouting, and sprouts exhibited lumens (Lower Right). Culture of EPCs and OECs on top of gels containing carrier beads with adherent ECs led to significant migration of the ECs toward the EPCs and OECs (Lower Center).

demonstrates cells on the top of the gel, with the bead on a different focal plane (indicating that cells in the main image

2.2 Scaffold

Next it was studied the capacity of the scaffolds to maintain cell viability,

proliferation and outward migration. This study was performed firstly in vitro (Fig.8).

To do so, the scaffolds containing the cells were embembed in collagen gel, and it was

quantified the migration of cells through that gel. After 72h of cell feeding, very few

OEC migrated out of the alginate scaffolds that did not contained RGD coupled.

Coupling the scaffold with RGD but not VEGF, led to an increase of the mobilization of

cells. And inclusion of specifically, VEGF121 led to even higher cell migration out of

scaffold. 60% of the cells that migrated out from scaffolds presenting VEGF121 were

viable (Fig.8).

2.3 Cotransplantation of EPC and OEC Enhances Neovascularization in Vivo.

Cotransplantation of EPC and OEC was done next to investigate to known

whether these two types of cell populations could together in vivo reproduce

neovascularization. It was used a bolus infusion of EPC and OEC as a control test. In

this case limbs with no necrosis (Fig.9). Animals treated with a bolus injection of both

cell types demonstrated a marginal recovery of regional blood flow (Fig.10). The use of

both EPC and OEC together with scaffolds containing VEGF121 induced a 2-fold

increase in vessel density, comparing to the control test. Animals treated with scaffolds

delivering cells and VEGF121 showed a marked increase in blood flow over time

(Fig.10). The necrosis of toes and foot was decreased when using a cotransplantation of

Fig.8 – On the left, Phase-contrast micrographs of OECs that have migrated out from scaffolds that contain no VEGF (blank), VEGF121, or VEGF165 and populated the surrounding tissue mimic (collagen gel) after 72 h. On the right, Cell migration assays. Diagram of the approach used to investigate the cell migration out of scaffolds.

OEC and EPC, and 30% of the mice used in this test revealed normal limbs after 6

weeks (Fig.9). Finally, a histologic analysis revealed that animals transplanted with

scaffolds with only EPC showed significant levels of adipose tissue, while animals that

took the transplantation of EPC and OEC revelealed normal tissue organization.

whereas animals treated with codelivery of EPCs and OECs by using implantable

scaffolds revealed normal tissue organization.

Fig.9 - Limbs with no treatment (blank scaffold), demonstrated precocious and rapid limb necrosis (_3 days) (left-most column), and no perfusion images were obtained. For other conditions, hindlimbs were maintained over time, and perfusion images could be obtained. In all of these conditions, scaffolds presenting RGD ligands and VEGF121 were used. The normal baseline (before) perfusion was immediately reduced after unilateral femoral artery ligation (after), and subsequent recovery was tracked as a function of time postsurgery.

Fig.10 - Quantification of hindlimb perfusion for the conditions, including bolus injection of EPC and OEC (inverted filled triangle), EPC transplantation with scaffolds (open square), OEC transplantation with scaffolds (open triangle), and EPC and OEC combined transplantation on scaffolds (filled circle) in SCID mice.

3. Discussion

After this work it is notable the application of EPC in the treatment of ischemia

and more broadly in regenerative medicine. These EPC are delivered by using an

appropriate material that provides cell support and stabilization. Observing the results

obtained, it is possible to say that this approach has many advantages in the treatment of

ischemic murine hindlimb musculature.

The polysaccharide used, alginate by itself does not mediate cell adhesion, so it

was covalently binded with other proteins (RGD and VEGF), by doing so it was

obtained a material that supported cells and at the same time allowed them to migrate

outwards the desired area of the problem. So, the major demonstration of this work is

that, coupling of an appropriate density of adhesion ligands to the polymer chains

dramatically increased OEC outward migration.

Cotransplantation of both EPC and OEC showed an increased

neovascularization of ischemic muscle tissue. The system used to deliver these cells

makes possible therapeutic angiogenesis, reversal of ischemia, and prevention of

necrosis and autoamputation. The results found here show that the success of these

therapies can be increased by controlling the delivery of cells in a manner that facilitates

the integration of EPC and OEC with the native cells populations.

This approach can be use to treat cardiac infarction, and other situations in

which neovascularization is lacking More broadly, this may provide a core technology

for the entire field of regenerative medicine, because of the need to create new vascular

beds in most situations of regeneration and tissue engineering (Johnson PC, 2007).

A more specific analysis, allows us to observe that OEC interact directly with

EC, supporting new blood vessels formation. Also, OEC and EPC secrete many

angiogenic factors, and likely this will increase EC migration when OEC and EPC are

cocultured. This finding, together with the finding that transplantation of OEC alon

increased the density of cessels in ischemic tissue, suggests that OEC are also important

for promoting host angiogenesis.

There are currently, many groups doing research in this area of cell delivery

systems. This approach may be broadly used to solve some of the problems associated

with current vascular cell-based therapies. Certainly, progenitor cells have lots of

potential and their fate inside the body and success treatment of ischemic tissue can be

controlled by the use of specifically designed delivery biomaterials.

4. Other applications of tissue engineering and future visions

Some molecules and drugs, have been introduced in the human body through

conventional delivery systems consisting in oral, intravenous and other ways. However,

conventional molecule delivery systems rely on the body to transport drugs, so it is a

passive system. Lately, nanotechnologies have been used for the creation of smart

nanomaterials, that can automatically deliver molecules to the desired area, as for

example, the central nervous system. Some of the example materials are:

Fig.12 - Main components of a bone repair system.

● Superparamagnetic iron oxide nanoparticles (SPION), that consist of inorganic

spheres with 10nm diameter coated either with organic or inorganic coatings, this to

improve biocompatibility and add functional groups. This can be a novel approach for

the local treatment of arthritis. As

so it is possible to use poly(lactic-

co-glycolic acid) PLGA

microparticles co-encapsulating the

anti-inflammatory drug

dexamethasone acetate and SPION

as intra-articular drug delivery

systems. Using this technology, the drug is gradually released, avoiding the formation

of crystals in the joint. Moreover, due to the magnetic nature of SPIONs, the

microparticles could be retained in the joint with an external magnet, thus reducing their

clearance from the joint (Fig. 11).

● ES cells are being used for the tissue engineering of blood vessels since

cardiovascular disease remains the leading cause of death in western countries and often

requires vascular reconstruction. To date, tissue engineered blood vessels (TEBVs)

could be successfully constructed in vitro, and are being used to repair vascular defects

in animal models. Must because of the complete study not only of how cells

differentiate and proliferate but also, how these cells will interact with the biomaterial

that supports cell growth, migration, differentiation and secretion of extracellular matrix

(ECM) proteins.

● In the last years, it has been directed many attention for the development of bone

substitutes based on osteoinductive growth factors incorporated in an optimal delivery

system. Ideally, this system should

be biocompatible, biodegradable,

and should preserve the growth

factors active and prevent the

excessive diffusion of the same

growth factors from the site of

application.

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