3.regenerative Med89

REGENERATIVE MEDICINE
1. Introduction
The tissue repair process is a complex cascade of biological events controlled by numerous
cytokines and growth factors that provides local signals at sites of injury. Just after a tissue injury
occurs, a large number of intercellular and intracellular pathways are activated and coordinated
[1]
with the aim of restoring tissue integrity and homeostasis . Cellular and humeral components,
the inflammatory pathways and the blood coagulation cascade are activated in addition.
A wide range of cells from the injured tissue and adjacent locations undergo marked changes
[2,3]
in gene expression and phenotype, leading to cell proliferation, differentiation and migration .
The development of a new microvasculature and microcirculation is also critical for the
homeostasis and correct tissue repair and regeneration. In fact, the absence of a suitable
vasculature transporting oxygen, nutrients, soluble growth factors and biologically active proteins
and numerous cell types to the injured tissue would imply the degeneration of the latter.
One pivotal discovery that has fuel the research in regenerative medicine and tissue
engineering has been the role that cytokine and growth factors play in the process of tissue repair
[5,6]
. These molecules provide signals at local injury sites, regulating the mechanisms and
pathways that govern wound healing and tissue regeneration [7]. Determining the roles that growth
factors play in tissue repair and regeneration is as important as designing, developing and
applying suitable formulations that release them with a spatiotemporal control. This is especially
important as providing the injured tissue a milieu of biological signals may be desirable for the
[8–11]
functional and accelerated repair of the tissue .This knowledge has stimulated the rapid
progress of tissue engineering. This field aims at repairing and restoring damaged tissue function
employing three fundamental ―tools‖, namely cells, scaffolds and growth factors. [12,13].
The success of this approach relies on the delicate and dynamic interplay among these three
[14,15]
components . It has been reported that future generation of scaffolds will have to provide
adequate mechanical and structural support, be biocompatible and bio-resorbable at a controllable
degradation and resorption rate as well as actively guide and control cell attachment, migration,
proliferation and differentiation. This may be achieved if the functions of scaffold are extended to
supply biological signals able to guide and direct cell function through a combination of cue
exposition and cytokine and growth factor controlled delivery [16].
In this review, we summarize the most interesting delivery technologies designed to permit
the local and controlled spatiotemporal release of growth factors for tissue repair and regeneration.
Especially, the progress that has been accomplished in the field of endogenous regenerative
medicine will be described in detail. The use of patient's own growth factors and scaffolds for
MALIBA PHARMACY COLLEGE Page 1

regenerative medicine will be highlighted, as some of the most interesting therapeutic applications
of this technology including the treatment of chronic ulcers, bone and soft tissue regeneration in
oral and maxillofacial surgery and oral implantology, the treatment of musculoskeletal injuries
and disorders, and the development of tissue-engineered approaches among others.

2. HISTORY:-
While transplant medicine made rapid progress, cell biologists were reporting similarly
important Advances, although the clinical significance of these had yet to be identified. From
initial research on cell and tissue culture back in the 1960s, where scientists were looking at ways
of keeping pieces of tissue alive outside of the body, work progressed to cell biology, where
different cell types were purified out of tissue samples to produce monolayer cultures of a single
cell type. Once scientists were able to do this, and began to understand what different cell types
made, the role of different metabolites and signals and how each cell behaved, tissue biology
emerged. Researchers started to look at ‗organotypic cultures‘, whereby two or more cell types
were grown together and their interactions examined.
 From there, it was a short intellectual step to tissue engineering, a term introduced by
YC Fung of the University of California, San Diego (CA, USA), in 1985 [17].
 Definitions of tissue engineering vary. In 1993, Langer and Vacanti described it as ‗an
interdisciplinary field that applies the principles of engineering and life sciences toward
the development of biological substitutes that restore, maintain, or Improve tissue function
or a whole organ‘ [18].
 MacArthur and Oreffo defined tissue engineering as ‗understanding the principles of tissue
[19]
growth, and applying this to produce functional replacement tissue for clinical use‘ .
Tissue engineering, subsequently identified as a subset of the overarching field of
regenerative medicine, was in effect born in Massachusetts as a result of that serendipitous
combination of circumstances that sometimes occurs in science. Much of the initial work in
the field began in four independent laboratories at the Massachusetts Institute of
Technology (MIT) in Cambridge (MA, USA), run respectively by Green, Bell, annas and
Vacanti (who collaborated with Naughton out of the Hunter College School of Health
Sciences, NY, USA) [20].
Each laboratory pursued a somewhat different strategy and each strategy eventually resulted in
approved medical products that are available today.
Harvard Medical School in Boston (MA, USA), WL Chick was working to develop a bio-
artificial pancreas [21]. His alternative approach, to encapsulate islet cells in order to isolate them
from immune attack, led to a variety of bio-hybrid organ projects some of which are showing
clinical promise.

[22]
By 1993, the first major review publication on tissue engineering appeared in Science
and, in 1998, the US FDA granted its first approval for an allogeneic tissue engineered product in
the form of Apligraf®, which was described originally as a living skin equivalent by
Organogenesis, a company spawned by Eugene Bell‘s laboratory. The launch of Apligraf ®
followed the autologous burns treatment, Epicel® which originated from Green‘s laboratory and
was, at first, unregulated by the FDA and treated as a tissue graft. Epicel® was originally
commercialized by Biosurface Technology and later by Genzyme Biosurgery after its purchase of
Green‘s company.
These products were followed by Dermagraft® produced by Advanced Tissue Sciences

(ATS) and commercialized through a joint venture with Smith and Nephew (ATS was renamed
from Marrowtec, a company formed by Gail and Brian Naughton to develop bone marrow feeder
cultures on 3D nylon meshes) [23]. Yannas‘ laboratory focused on the development of a cellular
scaffolds that would stimulate the patient‘s cells to repopulate and regenerate tissue. This led to
the development of the collagen/glycosaminoglycan sponge covered with a silicone membrane
called ‗Integra Dermal Regeneration Template‘, which was approved in 2002 for the treatment of
severe burns and commercialized by Integra Life Sciences. Although each of these therapies has
been important in its own way, rather like monoclonal antibodies a decade earlier and genetic
engineering a decade before that, initial product flow has been a small trickle rather than a mighty
deluge.

3. WHAT IS TISSUE ENGINEERING ?
Figure 1:- Tissue Engineering
 Tissue Engineering was postulated by Langer and Vacanti in 1993 as:

An interdisciplinary field that applies the principles of engineering and the life sciences
toward the development of biological substitutes that restore maintain, or improve the tissue
function.[24]‖
Many other, more or less similar definitions of tissue engineering can be found in the
literature. Being a relatively new field, tissue engineering, is not always clearly defined, and may
span from decellularised extracellular matrices, to exclusively cellular Implants or non-
biodegradable biomaterial scaffolds. Some examples of these definitions are:-
 The process of creating living, physiological, three-dimensional tissues and organs
utilizing specific combinations of cells, cell scaffolds, and cell signals, both chemical
and mechanical.[25]
 Some combination of cells, scaffold material, and bioactive peptides used to guide the
[26]
repair or formation of tissue.
 The three-dimensional assembly over time of vital tissues/organs by a process
Involving cells, signals and extracellular matrix.[27]
 The field of tissue engineering exploits living cells in a variety of ways to

Restore, maintain, or enhance tissues and organs[28]‖

 Products or processes that

(1) Combine living cells with biomaterials ,
(2) utilize living cells as therapeutic or diagnostic reagents,
(3) Generate tissues in vitro for therapeutic implantation, and
(4) Provide materials or technology to enable any of these approaches.[29]‖
Figure 2:-Cartilage regeneration Figure 3:-Artificial skin
Tissue engineering is a revolutionary addition to the therapeutic armamentariumum of

medicine .The intrinsic problem of transplantation is the chronic shortage of tissue organs. Tissue
engineering allows the hope of a regular creation of spare parts of the human body. Tissue
engineering is a most significant approach to reconstruct, replace, or repair organs in a way that
could not be foreseen 25 years ago.[30]
Reconstructive surgery: In 1970s the development of microsurgery allowed distant tissue
transfer and re -implantation (vascular grafts and prosthetic articulation). Tissue engineering is
remarkably multidisciplinary, bringing together cell and molecular biologists, biochemists,
engineers, pharmacologists, physicians, .etc.
Tissue engineering does open radically new chapter in reconstructive medicine, for it is now
deemed possible to reconstruct in the laboratory human living tissues and organs for in vivo, ex-
vivo, and even in vitro applications.
The aim of Tissue engineering is to obtain grafts for in vivo applications. The biological and
mechanical functions are of utmost importance.
 Biological functions: cell therapy
 Mechanical functions: tissue templates.
Tissue-engineered substitutes are three-dimensional reconstructions that can be implanted into
the human body, leading to rapid host implantation and acceptance.

3.1 Approaches to tissue-engineering substitutes [30]

1. Seeding of cells into various gels (collagen gels, fibrin and other component) Eugene Bell. It
was shown that the incorporation within the depth of these matrices with various types of cells
was possible.
The true integration of cells into gels allowed them to reorganize the surrounding matrix.
Drawback: Week mechanical resistance of the obtained substitutes. The structural integrity
may be sufficient for skin, but not so for substitutes in vascular or orthopaedic systems.
This problem has been addressed with glycation and magnetic alligement of the collagen fibers.
2. The seeding of cells into scaffolds:
The cell thrives in the porous material and secretes various amount of extracellular matrix
depending on their nature. (Developed by Robert Langer’s group MIT).
Sponge-like structures from mainly collagenous materials.
There have been countless modifications and additions to the different types of synthetic materials
used over the last decade in this approach.
The obvious advantage of the scaffold approach is the immediate creation of a three-
dimensional structure that already has significant structural properties.
Drawback: The intrinsic nature of most of these polymers, which are suture materials,
entails slow degradation with an ensuing lowering of pH of surrounding tissues. This leads
to a slow but rather protracted low-level inflammatory process.
Many groups are therefore attempting to alter the chemical nature of these biomaterials to
inhibit this inflammatory process.

3. LEOX group (Quebec, Canada):
In this approach various types of cells, mostly of mesencymal origin, are grown in such a
fashion within a culture flask that they literally embedded themselves in their very oven
extracellular matrix.
Among many factors, the addition of sodium ascorbate allows the significant appearance
of the various components of the extracellular matrix.
These sheets are than either stacked or rolled to obtain various tissue substitutes.
The re-creation of a totally biological vascular substitute.
The main advantage: The absence of extraneous collagens and any synthetic material.
Drawback: This approach is time consuming.
There are approximately 500,000 surgical procedures performed every year in the U.S. which
require bone substitutes. Currently available bone substitutes, including autografts, allografts ,
and synthetic materials, are the most implanted materials second only to transfused blood
products.
3.2 Autograft:-
A graft or portion of living tissue, taken from one part of the body and placed in another site on
the same individual.
3.3 Allograft:-
Grafts between two or more individuals allogenic (genetically different although belonging to or
obtained from the same species) at one or more loci. A bone graft is a piece of bone
transplanted to another part of the skeleton where it is needed to improve function or strengthen
the structure of the area. Sometimes a bone graft is taken from a cadaver, but usually it is
harvested from the patient for which it will be used. Bone grafts are typically harvested from
the patient's iliac crest (top of the hip bone), ribs, or fibula in the lower leg. This can be quite
painful and the complication rate can be high. Approximately 40% of spine fusion patients
complain of pain at the harvest site for as long as five years after surgery.

3.4 Why we need Tissue Engineering [30]

 Fewer livers available for transplant than there are patients waiting for new livers.
 A strategy for construction of the organ must be developed.
 Tissue engineering holds the promise of producing better organs for transplant
Using tissue engineering techniques and gene therapy it may be possible to correct many
otherwise incurable genetic defects.
 Artificial tissues can revolutionize healthcare by providing a supply of soft and hard
Connective tissues on demand.
 Other applications include replacement of lost skin; replacement or repair of defective or
Damaged bones, cartilage, connective tissue, or intervertebral discs; replacement of worn and
poorly functioning tissues (i.e. aged muscles or corneas); replacement of damaged blood
vessels; and restoration of cells that produce critical enzymes, hormones, and other
metabolites.
 Benefits
 Cost reduction in Expensive treatments.
 Dramatic improvements in Treatment outcomes Improved quality of life for patients.
3.5 PROCESS OF TISSUE ENGINEERING
(1) Start building material (e.g., extracellular matrix, biodegradable polymer),
(2) Shape it as needed.
(3) Seed it with living cells.
(4) Bathe it with growth factors.
(5) Cells multiply & fill up the scaffold & grow into three-dimensional tissue.
(6) Implanted in the body.
(7) Cells recreate their intended tissue functions.
(8) Blood vessels attach themselves to the new tissue.
(9) The scaffold dissolves..
(10) The newly grown tissue eventually blends in with its surroundings.

4. TYPE OF STEM CELLS USED IN THE TISSUE ENGINEERING:-
Figure 6:- Cell sources
Figure 7:-Cell differentiation

4.1 Sources of Stem Cells:-

1. Embryonic stem cells - are harvested from the inner cell mass of the blastocyst seven to
ten days after fertilization.
2. Fetal stem cells - are taken from the germline tissues that will make up the gonads of
aborted fetuses.
3. Umbilical cord stem cells - Umbilical cord blood contains stem cells similar to those
found in bone marrow.
4. Placenta derived stem cells - up to ten times as many stem cells can be harvested from a
placenta as from cord blood.
5. Adult stem cells - Many adult tissues contain stem cells that can be isolated.
4.2 Tissues:-
 Tissue is a cellular organizational level intermediate between cells and a complete organism.
Hence, a tissue is an ensemble of cells, not necessarily identical, but from the same origin,
that together carry out a specific function. Organs are then formed by the functional
grouping together of multiple tissues.
 The study of tissue is known as histology or, in connection with disease, histopathology.
 The classical tools for studying tissues are the paraffin block in which tissue is embedded
and then sectioned, the histological stain, and the optical microscope.

5. What is Regenerative Medicine?

Regenerative medicine is an applied field of tissue engineering that holds the realistic
promise of regenerating damaged tissue in vivo (in the living body)and externally creating tissue
for life available for implantation .though research and product developed from this field
previously untreatable disease will become easily and routinely cured.
5.1 How Regenerative Medicine works:-

Regenerative medicine is the application of tissue science, tissue engineering and related
biological and engineering principal that restores the structure and functions damaged tissue and
organ. This new field encompasses many novel approaches to treatment of disease and restoration
of biological functions through following methods:
 Using therapies that promote the body‘s to autonomously regenerate damaged tissues
 Using tissue engineering implants to promote regeneration
 Direct transplantation of healthy tissue into damaged environments
Collectively, the treatment allows two substantial advances is the potential to in vivo (in the
living body regenerate currently irreparably damaged tissue so that they return to full
functionality. The second advance is to able to produce tissues in vitro (in the laboratory0to be
used for transplantation purpose when regeneration is not possible. This technology has the
potential to cure disease from diabetes (through regeneration of islets‘) to the repair of cancerous
tissue (by replacing the removed cancerous tissue with externally grown healthy tissue).by
creating these ―tissue for life‖ regenerative medicine treatments will undoubtedly lead to a
tremendous improvement in quality of life and health care.
5.2 Why do we need Regenerative Medicine?

Regenerative medicine is a revolutionary approach that focuses on curing conditions as
opposed to treating them. Regenerative medicine empowers doctors with the ability to replace
damaged tissue in patient with healthy organic tissue that is accepted and function like (and in
some cases, is) the body‘s own. These therapies will cure a variety of diseases ranging from
diabetes to cancer. Regenerative medicine will lead to improved patient care while eliminating the
cost of treatment such as insulin injections or dialysis.

5.3 VARIOUS FIELD RELETATED TO REGENERATIVE MEDICINE:-

Figure 6:-Cell therapy

 Transplanted cells:-
 Mature, functional cells
 Modified cell human cells
 Tran differentiated own patient‘s cells
 Non-human cells (XENOTRANSPLANTATION)
 Stem cells (autologous or allogeneic)
5.4 Cell therapy challenges:-

 Directing cells to the proper place
 Integrating cells with the patient‘s own tissue
 Overcoming the phenomenon of tissue rejection
5.5 TISSUE ENGINEERING APPROACH:-
Tissue Engineering Process

6. What is Scaffold-cell?
The scaffold is three-dimensional and is composed of hollow or solid fibers of a
biocompatible, synthetic polymer which is biodegradable or non-biodegradable. The fibers of the
scaffold may have a branched configuration extending outwardly from a central stem. Fibers of
the scaffold are spaced apart such that the maximum distance over which diffusion of nutrients
and gases must occur through a mass of cells attached to the fibers is between 200 and 300
microns. The diffusion provides free exchange of nutrients, gases and waste to and from cells
proliferating throughout the scaffold in an amount effective to maintain cell viability throughout
the scaffold in the absence of vascularization. Cells derived from vascularized organ tissue are
attached in vitro to the surface of the fibers uniformly throughout the scaffold in an amount
effective to produce functional vascularized organ tissue in vivo, the cells are grown on the
scaffold in a nutrient solution in vitro to form the cell-scaffold composition which is implanted in
a host at a location having adequate vascularization to allow growth of blood vessels into the cell-
scaffold composition. Growth factors, compounds stimulating angiogenesis and
immunomodulators may be provided in the cell-scaffold composition and the fibers may have a
coating to enhance cell attachment. Combinations of cell-scaffold compositions containing
different cell populations may be implanted.
Figure 7:-Scaffold cell delivery
Figure 8:-Process of scaffold seeding

6.1 Scaffolds for Tissue Engineering and Regenerative medicine:-

For most of the decades of the 20th century, biomaterials have played a critical role in
enabling the fabrication of a large number and wide variety of medical implants. Except for a
few examples, however, these were permanent devices meant to fix or replace the function of
tissues and organs. Stainless steel devices were developed for the fixation of fractures and to
fix allografts to host bone. Implants fashioned from metallic, ceramic and polymeric materials
facilitated life-saving procedures in many patients (eg vascular prostheses and artificial heart
valves) and profoundly improved the quality of the lives of other individuals (eg joint
replacement prostheses). Despite these remarkable successes, the new roles for biomaterials in
medicine will likely exceed these achievements.
The new roles include the use of porous, absorbable biomaterial (sponge-like) scaffolds in
tissue engineering, regenerative medicine, and gene therapy. Scaffolds for engineering bone
and the soft tissues have been synthesized from an array of synthetic and natural calcium
phosphates and myriad synthetic (eg. polylactic acid and polyglycolic acid) and natural (eg
collagen and fibrin) polymers. Scaffolds for engineering tissue in vitro, or to be used as
implants to facilitate regeneration in vivo, need to have a microstructure and chemical
composition able to accommodate cells and their functions. In this regard a porous structure is
generally necessary. The required porosity and pore diameter, pore distribution, and pore
orientation, might be expected to vary with tissue type. The chemical composition of the
matrix is important with respect to its influence on cell adhesion and the phenotyp ic
expression of the infiltrating cells. Moreover, because the objective is the regeneration of the
original tissue, the scaffold needs to be absorbable.
The degradation rate of the material generally may be determined based on the rate of
new tissue formation and the normal period for remodelling of the tissue at the site of
implantation. Of course, it is important to consider the effects of moieties released during
degradation of the matrix on the host and regenerating tissue. Finally, the mechanical
properties of the biomaterial employed as a scaffold for tissue engineering are important for
providing temporary support of applied loading in vivo during the regeneration process and for
resisting the contractile forces that may be exerted by the seeded cells prior to implantation
and by cells infiltrating the scaffold in vivo.

Figure 9:-Microscopic appearance of scaffold cell
6.1.1 Scaffold:-
• Allow cell attachment and migration .
• Deliver and retain cells and biochemical factors .
• Enable diffusion of vital cell nutrients and expressed products ..
• Exert certain mechanical and biological influences to modify cell behaviour.

Figure 8:-Demonstration of use of scaffold cell
6.1 Bio-material used in scaffold:-
Material Relevant features and application

Naturally-derived Collagen-based scaffolds Soft tissue repair
Hyaluronic acid (HA) and derivatives Cell differentiation
Collagen-HA gels Capillary engineering
Chitosan Dermis engineering
Fibrin Vascularized adipose tissue
Gelatin Regeneration of skin, cartilage
Patterning of cell growth
Control of vascular sprouting
Chitosan microsphere-integrated scaffold
Cartilage engineering
Vessel engineering
Release of fibroblasts
Trachea engineering
Bone engineering
Vascular engineering
Synthetic Poly(glycolic acid) (PGA) Musculoskeletal tissue

Poly(lactic acid) (PLA) engineering
Polylactide-co-glycolide (PLGA) Cartilage regeneration
Poly(?-caprolactone) (PCL) Fibrovascular engineering
Polyethylene glycol (PEG) Skin engineering
Oligo(poly(ethylene glycol) fumarate) (OPF) Bone formation
GF release from gelatin microspheres
Inorganic Tricalciumphosphate (TCP) Bone substitute
Hydroxyapatite (HA)
Semi-synthetic Cross-linked thiolated HA Neurite growth and support
Esterified hyaluronan (HYAFF® derivatives) Vocal fold repair

6.3 Roles of a Scaffold in Tissue engineering and Regenerative medicine [28]:-

There are many roles that a scaffold can play in the tissue regeneration process:
 The scaffold can serve as a framework to support cell migration into the defect from
surrounding tissues; especially important when a fibrin clot is absent.
 Before it is absorbed, a scaffold can serve as a matrix for endogenous or exogenous cell
adhesion, and can facilitate/regulate certain cell processes including mitosis, synthesis, and
migration. This may be mediated by ligands for cell receptors (integrins), on the biomaterial
and/or the biomaterial may selectively adsorb cell adhesion proteins.
 The scaffold may serve as a delivery vehicle for exogenous cells, growth factors, and
genes. This activity is enabled by a large surface area for attachment and the possible
control of the density of the agents (i.e. agents/unit volume).
 The scaffold may structurally reinforce the defect to maintain the shape of the defect and
prevent distortion of surrounding tissue. – The scaffold can serve as a barrier to prevent the
infiltration of surrounding tissue that may impede the process of regeneration.

7. Application of Tissue Engineering and Regenerative Medicine

7.1 Wounds and Burns Care[29-32]:-
®
Product name: - Integra
Figure 9:-Integra wound dressing

DISCRIPATION: -
Wounds and burns that penetrate deep into the skin present major clinical
problems for patients and healthcare professionals alike. Transplantation of the patient‘s own skin by
way of a split-thickness skin graft remains the ‗gold standard‘ in the treatment of major skin loss.
However, this incurs significant donor site morbidity and harvest sites are limited. Advances in tissue
engineering mean that there is now a limitless supply of skin through the development of innovative
products such as Integra®, a matrix designed to provide immediate wound closure and permanent
regeneration of the dermis.
 In the absence of underlying disease, almost every full-thickness wound will heal with minimal
intervention, however, the process can be enhanced by judicious wound management. The first
clinical decision to be made is whether to repair the wound or to allow it to heal by secondary
intention (Rivera and Spencer, 2009). There are four phases to the healing process:
1. The inflammatory phase involves bleeding, immediate narrowing of the blood vessels
and clot formation
2. The proliferative phase is where new skin cells and blood vessels are formed
3. The remodeling phase takes place after 2–3 weeks and involves the replacement of the
collagen layer
4. Finally, the skin forms a protective barrier (epithelial cells) between the outer environment
and the body.

 Advances in wound care have resulted in a vast range of products that can accelerate healing,
regenerate the dermis and reduce bacterial inflammation. Furthermore, in complex full-
®
thickness wounds, the use of skin substitutes such as Integra Dermal Regeneration Template
(Integra) (Figure 1) is gaining popularity. Dermal regeneration templates have been used for
three decades in the management and reconstruction of complicated wounds (Yannas et al,
1982)
7.2 Hair Stimulating Complex:-(HSC)[34]

DISCRIPATION: - HSC is a proprietary formulation of naturally secreted embryonic-like
proteins and growth factors. In addition to Wnt 7a, which is recognized to be critical in the
induction and maintenance of hair follicle growth, the complex contains a wide variety of factors
typically produced by embryonic cells and which are important to the hair cycle, such as
follistatin. While these embryonic-like materials are amplified under Histogen's unique
manufacturing conditions, undesirable proteins and growth factors, including Wnt 5a, which has
been shown to be associated with cancer, are eliminated.
Patients treated with Histogen's Hair Stimulating Complex (HSC)
3 months
Hair count + 22.4%

Terminal hair + 27.8%
Thickness + 23.9%
Hair Regrowth results after 3 months

Baseline results

7.3 Regenerated Airway[35]:_-

("In Breath" Bioreactor)
DISCRIPATION:-
Harvard Bioscience, Inc. (Nasdaq:HBIO), a global developer,manufacturer and marketer of a
broad range of specialized products used to advance life science research, today announced the
launch of its ORGANIZER™ Series Model 100 "In Breath" bioreactor. This bioreactor was used
to grow the bronchus used in the landmark clinical transplantation performed by Professor Paolo
Macchiarini of the Department of General Thoracic Surgery,Hospital Clinic, Barcelona, Spain.
Figure 10:-Regenerated airway
In this surgery Professor Macchiarini and colleagues removed a trachea from a human
cadaver, decellularized it (i.e., removed all cellular material leaving the extra cellular matrix
scaffold behind) and, using the bioreactor, recellularized the scaffold with adult stem cells
cultured from a biopsy of the patient's own tissue. After four days in the bioreactor the tissue
engineered airway was implanted in the patient replacing her defective left main bronchus. The
patient made a full recovery, showed a substantial improvement in quality of life and showed no
immune rejection of the new airway.

7.4 Treatment of blindness by corneal stem cells[36-43]:-
The eyeball is covered by the cornea — the eye‘s most important light-refracting structure
(Fig. 1a). The cornea produces the initial image and casts this onto the lens behind it.The clearness
of the cornea is essential to visual acuity and depends on both the integrity of the corneal
epithelium covering the eye‘s surface and a lack of blood vessels in the underlying support tissue
(the stroma). At its margins, the corneal epithelium is attached to the delicate mucous
(conjunctival) epithelium that covers the whites of the eye (sclera) and the internal part of the
eyelids. The narrow zone between the cornea and the conjunctiva is known as the limbus.
Experimental and clinical evidence indicates that the limbus is the source of corneal epithelial
stem cells in humans.The limbus can be destroyed by ocular burns or infection, causing corneal
stem-cell deficiency. But, in one of nature‘s remarkable efforts to repair tissues at all costs,
abnormal invasion by conjunctival cells provides the damaged cornea with a protective surface
layer . The consequences are dire, resulting in vascularization of the cornea, chronic
inflammation, stromal scarring and, ultimately, corneal opacity and loss of sight2.Allogeneic
corneal transplantations, which involve transplanting cornea from a genetically non-identical
donor, have to some extent been successful in restoring patients‘ vision.

Eventually, however, conjunctiva cells invade and replace the transplanted cornea. What‘s
more, two other factors make treating patients with ocular burns by corneal transplantation
problematic: the number of available donors is insufficient to meet demand, and the increasingly
popular corrective laser eye surgery often makes the cornea unsuitable for transplantation.
Pellegrini, De Luca and colleagues1 now report that limbal stem cells maintained in culture can be
a viable alternative source of cells for transplantation to treat burned human corneas.Stem-cell
transplantation is not a new concept.More than half a century ago, E. Donnall Thomas showed
that intravenous infusion of donor bone-marrow cells can repopulate the bone marrow and
produce new blood cells4;he later won a Nobel prize for this first demonstration of the use of stem
cells for regeneration of damaged or diseased tissues and organs. By the 1970s, physicians were
successfully performing bone-marrow transplants, which are now used to treat blood disorders
ranging from severe combined immunodeficiency to sicklecell anaemia to leukaemias, as well as
other cancers of the human immune system.
By the early 1980s, human skin stem cells were being cultured to make epidermal sheets to
repair the skin of badly burned patients. Pellegrini, De Luca and colleagues have been culturing
corneal stem cells from small biopsies of human limbal tissue for the past decade. The appreciable
similarities between limbal and epidermal cells allowed the researchers to adapt methods
developed for human epidermal stem-cell cultures. Cultured epidermalcell colonies can be
classified according to cell number and capacity for growth. The smallersized colonies generate
epidermal cells that stop growing over time. By contrast, the largersized colonies — referred to as
holoclones —display quintessential features of stem cells, namely long-term self-renewal and the
ability to regenerate tissue. This makes them suitablefor burn therapy.Pellegrini, De Luca and co-
workers7 discovered that human limbal cells cultured using a similar protocol also form small and
large colonies.Interestingly, only the limbal holoclones and not the smaller colonies expressed
p63,a transcription factor that is essential for theproliferative potential of epidermal stem cells8.In
the impressive accompanying clinical studies9,the researchers obtained limbal stem cells from the
healthy eye of 112 patients with ocular burns, cultivated them and then transplanted the cultured
cells onto the patients‘ damaged eye. After an extensive 10-year monitoring period, the authors
now report1 permanent restoration of a transparent, self-renewing corneal epithelium in three-
quarters of the study patients (Fig. 1c).
Notably, 78% of the successful transplantations involved cultures in which p63-expressing
cells constituted more than 3% of the cells capable of forming colonies.These observations unveil
a direct correlation between the percentage of p63-positive corneal stem cells in a culture and their
transplantability.The correlation presents a powerful diagnostic tool for predicting whether any

given limbal culture is likely to be suitable for long-term transplantation.This work1 also offers
hope for exploring alternative sources of limbal stem cells to treatpatients who have suffered
severe injuries to both eyes, and who therefore lack limbal stem cells. Indeed, in the future it
might be possible to create corneal stem cells by culturing other cells from the patient — for
instance, skin stem cells — and then either directly inducing their transdifferentiation to limbal
cells, or transforming them first to an embryonic-stemcell-like state (induced pluripotent stem
cells,or iPS cells) before inducing their differentiation along the limbal lineage.
7.5 Heart valve tissue engineering[45-47]:-
Tissue engineering, the successful fabrication of autologous living cardiovascular

replacements similar to their native counterparts is supported by three main elements:(1)
autologous cells that resemble their native counterparts in phenotype and functionality,(2) a
temporary supporter matrix (biodegradable scaffold) which promotes tissue strength until the
extracellular matrix produced by the autologous cells guarantees functionality on its own, and
(3)culture conditions enabling tissue formation and maturation by in vitro conditions similar to a
physiological environment. Figure 1 summarises the concept of in vitro tissue engineering.
Concretely, in a first step cells are harvested from an autologous donor structure. After expansion
in vitro, cells are seeded onto a biodegradable heart valve scaffold, which should ideally be at
least 90% porous . Scaffolds could be fabricated from polymers and the use of these different
synthetic materials has already been broadly demonstrated for cardiovascular tissue

engineering.An overview about the most common scaffold types applied in cardiovascular tissue
engineering is given in table 1. After seeding, the constructs are cultured in nutrient media
supplemented with ascorbic acid and 10% foetal calf serum in a pulse duplicator in vitro system
(bioreactor) mimicking the in vivo environment. In order to improve cell migration, proliferation
and extracellular matrix production mechanical load by means of shear stress have been applied to
the seeded valves in pulsatile flow bioreactors .
There, the tissue formation takes place and after several days the constructs are ready for
implantation. This concept for in vitro heart valve tissue engineering was earlier applied in an
animal model using completely autologous tissue engineered heart valves based on polyglycolic
acid coated with poly-4-hydroxybutyrate (PGA/P4HB) (figure 2) starter matrices . In this ―proof
of principle‖ study trileaflet heart valve scaffolds were fabricated from PGA/P4HB bioabsorbable
polymers and sequentially seeded with autologous ovine myofibroblasts and endothelial cells. The
constructs were grown for 14 days in a pulse duplicator in vitro system under gradually increasing
flow and pressure conditions and implanted into a growing sheep model (n = 6 lambs; mean
weight at cell harvest 9 ± 2.8 kg). Echocardiography demonstrated mobile, functioning leaflets
without stenosis, thrombus or aneurysm. These autologous tissue engineered valves functioned up
to 5 months in vivo and resembled normal heart valves as to microstructure, mechanical properties
and extracellular matrix formation.
7.5.1 Human marrow stromal cells:-
Figure 11:- HEART VALVE

With regard to future routine clinical realization of the tissue engineering concept, human marrow
stromal cells are a promising cell source. In contrast to vascular cells, these cells can be obtained
without surgical interventions representing an easy-to-access cell source in a possible routine
clinical scenario. The usage of marrow stromal cells may offer several advantages in i) easy
collection by a simple bone marrow puncture avoiding the sacrifice of intact vascular structures,
ii) showing the potential to differentiate into multiple cell lineages, and iii) demonstrating unique
immunological characteristics allowing persistence in allogenic settings. Recently, human marrow
stromal cells have been used for the fabrication of trileaflet heart valves (figure 3) . Histology of
the tissue engineered valve leaflets revealed viable tissue organized in a layered fashion with
extracellular matrix proteins characteristic of heart valve tissue such as collagen I and III, and
glycosaminoglycans. However, the typical three layered structural composition of native valve
leaflets comprising a ventricularis, spongiosa and fibrosa layer was not achieved. The ultra-
structural analysis of the tissue engineered heart valves supported this observation demonstrating
cell elements typical of viable, secretionally active myofibroblasts such as actin/myosin filaments
as well as collagen fibrils and elastin. The quantitative extracellular matrix protein analysis
revealed values significantly lower compared to human native valve tissue.
7.6 BONE REPAIR:-

 Obtain patient‘s bone cells
o From the hip
 Seed onto scaffold
 Implant scaffold
o Use body as incubator
o All appropriate growth factors available
 Remove and implant in proper location
Figure 12:- Artificial bone (lower jaw)

7.7 Tooth Regeneration Using Adult Stem:-
Figure 13:-TOOTH REGENERATION
 Epithelial stem cells can be harvested from the palate while mesenchymal stem cells can
be obtained from either bone marrow or the pulp tissue of an extracted third molar
 Stem cells are isolated and multiplied in vitro by using appropriate supportive culture
media
 At the late cap or bell stage the tooth germ is transplanted into the jaw

7.9 Autologous Cultured Cartilage

Cartilage is the thin layer that covers the bones in joints such as knees and elbows, allowing
them to move smoothly over each other. In healthy adults this layer is around 2-3 mm thick, but it
becomes thinner with advancing age. As cartilage tissue does not naturally recover once damaged,
it is extremely difficult to treat with drugs or other therapies. Now, however, the latest tissue-
engineering technology means cartilage can be regenerated with cultured cartilage.
Figure 14:- Cartilage
Development:-
The treatment of damaged cartilage has long been one of the aims of orthopedic
surgery. Professor Mitsuo Ochi of Hiroshima University has taken small amounts of cartilage
from patients with cartilage damage and produced cultured cartilage, which is then implanted into
the defective area. The therapeutic technique he established is known as autologous cultured
cartilage transplantation. J-TEC was quick to take notice of this method and obtained the guidance
of Professor Ochi concerning his culture technique, in order to develop Japan's first ever cultured
cartilage.
Culture:-

An orthopedic surgeon carries out minimally invasive arthroscopic surgery (keyhole

surgery) to collect a small amount of cartilage from the knee. This cartilage is sent to J-TEC and
cultured after having been mixed with atelocollagen gel and shaped into a three-dimensional form.
During the culture period, which lasts about four weeks, the cartilage cells (chondrocytes)
proliferate and eventually reach a state closely resembling the properties of the original cartilage.
This method is known as three-dimensional culture, and it is outstanding for the fact that it
enables chondrocytes to be cultured while retaining their original properties.

8. What is the current state of regenerative medicine[49-50]?

Regenerative medicine to date has made a number of advances in the field of simple
tissues such as skin, bone, and cartilage. Progress toward more complex therapies has been limited
due to thereliance on private sector funding and a lack of understanding of fundamental tissue
interactions.Private sector funding has led to a product oriented approach that does not focus on
research or consider the fundamental science issues in regenerative medicine. Without this
fundamentalresearch, it will be exceedingly difficult for the regenerative medicine field to develop
more advanced tissues and organs. To accommodate new biomedical technology, the FDA has
created the Office of Combination Products and the Office of Cellular, Tissue and Gene
Therapies.
These new offices solve the problem of regulating complex combination products that
typically fell under two or more of the FDA‘s traditional offices. These changes help prepare the
FDA for regenerative medicine product regulation and product approvals.
8.1 Current technological accomplishments in regenerative medicine [49-50]

Regenerative medicine research is a new science, with a many new concepts being
researched and tested but few effective products are on the market. From a research standpoint,
several key methods and approaches have been established including: • Direction of cell
expansion and differentiation, which explains the processes of how tissues and organ grow •
Development of techniques for assembly of cells into large, three dimensional tissue-like
structures, which will lead to the physical creation of three dimensional organs
• Custom-designed biomaterials to serve as structural templates for tissue development,
which helps scientists build organs
• Automated bioreactor culture vessels, which allows scientists to mass produce cells and
tissues these techniques have allowed researchers to begin to understand at a very basic
level how cells and tissues function and interact. At a functional level, regenerative
medicine researchers have been able to grow heart arteries and create artificial blood in the
laboratory. Engineered bladders, ligaments and stem cell therapies are in various stages of
preclinical and clinical tests. However, the only FDA-approved and available products are
much simpler tissues, such as dermal and joint substitutes, and bone marrow for
orthopedics.Many skin substitutes have been used successfully ,but cartilage and bone
replacement techniques have been more difficult.16 As shown by the limited scope and
inconsistent performance of the products available, regenerative medicine is at a very early

stage of development, hampered by a lack of cohesive fundamental research to advance

the field. It is this research that is needed for the science of regenerative medicine to
realize its full potential of restoring even the most complex of tissues to full health.
8.2 Regulatory environment [49-50]

The FDA has begun working towards creating an environment that accelerates the
approval of combination products. Therapies are classified by the FDA as either a device,
biologic, or drug .Each classification has differing regulations and its own review Center
consisting of experts in a given field. Regenerative medicine products fall under some or all of
these classifications, requiring regulators to appropriately integrate different regulations to ensure
adequate product safety and effectiveness. In the past, this added complexity led to combination
products requiring longer approval times than standard products. To accommodate the
advancements of medicine, the FDA created its Office of Combination Products (OCP). The
Medical Device User Fee and Modernization Act of 2002 established this new office on
December 24, 2002. Under this law, the OCP is given the power to ensure the timely review of
drug-device, drug-biologic, and devicebiologic combination products.22 Examples of some
combination products that OCP has handled include:
• Dermagraft, which uses an absorbable scaffold to deliver collagen and other skin tissues to
wounds
• InFUSE Bone Graft/T-CAGE lumbar tapered fusion device, which is a treatment for a
degenerative disc disease by stabilizing the spacing in the spine and then forming new
bone • Antibiotic bone cement, used for fixating prosthesis to living bone23 These
products are all combinations of drug, biologic and devices. While these products are
considerably simpler than organ tissues, thus far the FDA has shown that it is prepared to
efficiently regulate regenerative medicine products as they become available. The FDA
has also established the Office of Cellular, Tissue and Gene Therapies (OCTGT).The
OCTGT consolidates a number of regulatory programs into a single entity with a single
expert staff. These consolidated groups include:
• Human tissues
• Cellular therapies
• Xenotransplantation
• Gene therapies

By creating these new offices for regulation, the FDA has begun to evolve into a more
nimble approval agency capable of handling the advances of the 21st century .To complement
these new offices, the FDA has also begun collaborating with various NIH Institutes and
participating in the Multi-Agency Tissue engineering Sciences Working Group to raise awareness
of FDA guidelines and procedures. Another innovative initiative the FDA has begun in
conjunction with the National Cancer Institute allows cancer researchers to link their
investigational new drug (IND) applications to the FDA. This program has the goal of reducing
process and submission times for researchers. If successful, this program will provide a model for
IND applications in other fields, including regenerative medicine. While the FDA has made great
strides towards embracing new technologies, the private sector still views the regulatory process
as somewhat difficult. FIRM will provide a cohesive framework whereby public and private
funding organizations will partner with the FDA very early in the development of regenerative
medicine products to facilitate transparency in the regulatory oversight process for these new
products. By partnering in this manner, FIRM will set a new standard for industry and FDA
cooperation that will lead to faster product approvals without sacrificing safety or efficacy.

9. What is the future of regenerative medicine technology [50]?

Despite the availability of some first generation products, researchers know very little
about the underlying science of regenerative medicine. In order to build complex tissues and
organs, scientists first must understand how tissues interact with each other. To achieve the
envisioned goals of regenerative medicine, a strong Federally-directed initiative is needed to
ensure that this fundamental research is realized.If a directed and well-funded research effort were
to begin, regenerative medicine could begin producing results within 5 years. At the 5-year mark,
complex skin, cartilage, bone, and blood vessel products would begin to reach markets.Within 10
years, organ patches that repair damaged tissues would potentially be available. Within 20 years,
full organ regeneration is a strong possibility .For these products to be regulated safely and
efficiently, the FDA must continue its recent admirable moves to embrace new technology. The
FDA has recognized the challenge of new medical technology and has worked to improve
outreach and education about the regulation process, created a new office for handling the
evaluation of combination products, and created sub-offices for handling tissue and genetic
therapies. This commitment to the future is encouraging and the FDA remains involved in
developments relatingto regenerative medicine research. Other countries have already embarked
upon national initiatives of their own with hopes of making regenerative medicine a reality for
themselves. Several members of the European Union (EU), including Great Britain, Germany ,and
Sweden, as well as Japan, China and Australia have all begun making strong national
commitments with hopes of achieving their own advances in regenerative medicine technology. It
is time for the U.S. to commit its own resources and work with these nations as a leading partner
in driving this technology forward.
9.1 Next steps for regenerative medicine [50]

Although regenerative medicine as a field has existed for over 10 years, surprisingly little
basic research has been done. In order for the field to 11advance, scientists must research
fundamental cellular relationships and develop techniques for cellular production and
preservation. In order to eventually realize the concept of tissues on demand, interim research
steps must be achieved. Examples of interim research goals that must be achieved include:
Understand the processes involved in mechanical signaling and cellular
mechanotransduction, which explains how cells and systems communicate with each other.

• Improve control of organogenesis, which is the control of tissue development

• Create tissues in vitro (in the laboratory) and then bringing these tissues in situ (in the
natural environment), which teaches scientists how to integrate laboratory grown cells into
actual living bodies
• Develop handling and storage procedures for regenerative medicine applications, in order
to effectively anage and preserve tissue supplies
• Increase scalability of engineered cells, that enables scientists to mass-produce engineered
cells ensuring enough heart, skin, pancreas, and other needed tissues are available
• Develop tissue quality assurance procedures, to ensure tissues are safe and consistent like
any mass- produced item
Despite 10 years of study in regenerative medicine, there has been a lack of directed
research. Although much has been learned on these subjects the field is still in an embryonic
stage. Without this fundamental research, the potential of fully engineered complex tissues will
never be realized. If regenerative medicine researchers and clinicians are able to gain a detailed
understanding of how cells interact with each other and how to mass-produce, preserve, catalogue,
and build these cells, they can then apply this knowledge towards developing tissue and organ
based therapies. One example of a complex regenerative medicine issue that scientists must solve
is the growth of vasculature in tissues and organs. These vascular tissues are blood vessels
responsible for transporting nutrients and waste through tissue. Due to a lack of understanding of
cellular interaction, scientists have not been successful in creating vasculature in tissues and
organs, limiting regenerative medicine products to ―two dimensional‖ materials, such as skin and
bone, which do not require vascular tissue upport. To achieve the promise of regenerative
medicine ,growing vascularized tissues is a necessary next step. To do this, scientists must gain a
better understanding of tissue interactions and scaffold technology. Once scientists understand
these concepts, they will be able to apply this knowledge and create more advanced tissue
systems. Ultimately, the application of knowledge of cellular interactions and tissue growth will
culminate in two branches of regenerative medicine research, in vivo cell based therapy and in
vitro grown tissues and organs. Cell-based therapy focuses on cellular treatments that lead to
regeneration by having the body ―gather‖ the necessary reparative cells and bring them to the
damaged site. The second branch is the in vitro growth of tissues and organs that are then
implanted within the body, either to prompt regeneration or to replace damaged tissue.30 Each
branch of research offers substantial advances over current medicine, and discoveries from one
branch may be directly applicable to the other. Both of these research branches are vital for fully

realizing all of the potential therapies of regenerative medicine. For example, preliminary research
has shown that spinal cord regeneration through implantation of seeded scaffolds is feasible.31
Seed-driven regeneration is a form of cell-driven therapy that will not require transplantation of a
new tissue or organ. On the other hand, it is believed that treatment of a cancerous lung would
require removing the lung and replacing it with a laboratory grown healthy lung. In the case of
cancer, seeding the lung for regeneration would not work, as the cancer would still be present.
These two different applications of regenerative medicine demonstrate why it is essential to
research both cellular therapies and full organ growth techniques to maximize the potential of
regenerative medicine. It may be possible to use regenerative medicine to cure a diseased lung
without removing it, or to cure spinal injuries through neural cell transplants. What is most
important is that these two branches be investigated fully: findings from both will further our
knowledge of regenerative medicine .Fulfilling these goals will create a foundation for future
regenerative medicine products and work .A cohesive effort focused on advancing the science and
the field as a whole is essential.
With a cohesive Government initiative and appropriate funding, within 20 years
regenerative medicine will be the standard of care for replacing all tissue/organ systems in the
body in addition to extensive industrial use for pharmaceutical testing. The ultimate goal at the
end of 20 years is to have real time mass customization of tissues on demand, in vivo. During
those 20 years, as our knowledge of tissues grows, it is reasonable to expect to see treatments
discovered along the way, roughly at the 5, 10 and 20 year marks. In 5 years the following
milestones are hoped for:
• Develop multiple applications for skin, cartilage, bone, blood vessel, and some
urological products
• Develop insurance reimbursable regenerative therapies • Establish standards for FDA
regenerative medicine therapy product approvals
• Solve cell sourcing issues, giving researchers access to the materials they need to
design new therapies
• Establish cost-effective means of production, paving the way for future products •
Establish specialized cell banks for tissue storage, allowing storage of viable ―off the
shelf‖ products

In 10 years, effective regenerative medicine therapies will be available for patient care and
industrial research and development purposes. At this time, the following may be achieved:
• Further understand stem cell and progenitor cell biology
• Engineer smart degradable biocompatible scaffolding
• Develop microfabrication and nanofabrication technologies to produce tissues with
their own complete vascular circulation
• Develop complex organ patches, that could repair damaged pieces of the heart or other
organs
 Ultimately, within 20 years the full benefits of regenerative medicine therapies will be
reached. Some of the applications of regenerative medicine could be:
• Harness regenerative medicine materials to produce in situ regeneration of diseased and
damaged structures in many areas of the body
• Regenerate most damaged tissues and organs either in vivo or through implanted
regeneration therapies
• Produce in vitro sophisticated 3-D tissues and organs that cannot be regenerated through in
vivo techniques, such as an entire heart or lung Without a Federal initiative supporting
this research, this timeline could extend over the next 40 to 50 years. Considering the
many economic and health advances this technology may bring, it is absolutely vital that
regenerative medicine advance as quickly as possible.

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REGENERATIVE MEDICINE

MALIBA PHARMACY COLLEGE Page 1

MALIBA PHARMACY COLLEGE Page 2

MALIBA PHARMACY COLLEGE Page 3

These products were followed by Dermagraft® produced by Advanced Tissue Sciences

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3. WHAT IS TISSUE ENGINEERING ?

Figure 1:- Tissue Engineering

 Tissue Engineering was postulated by Langer and Vacanti in 1993 as:

 The field of tissue engineering exploits living cells in a variety of ways to

MALIBA PHARMACY COLLEGE Page 5

 Products or processes that

Figure 2:-Cartilage regeneration Figure 3:-Artificial skin

Tissue engineering is a revolutionary addition to the therapeutic armamentariumum of

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3.1 Approaches to tissue-engineering substitutes [30]

2. The seeding of cells into scaffolds:

Sponge-like structures from mainly collagenous materials.

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3. LEOX group (Quebec, Canada):

The re-creation of a totally biological vascular substitute.

Drawback: This approach is time consuming.

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3.4 Why we need Tissue Engineering [30]

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4. TYPE OF STEM CELLS USED IN THE TISSUE ENGINEERING:-

Figure 6:- Cell sources

Figure 7:-Cell differentiation

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4.1 Sources of Stem Cells:-

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5. What is Regenerative Medicine?

5.1 How Regenerative Medicine works:-

5.2 Why do we need Regenerative Medicine?

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5.3 VARIOUS FIELD RELETATED TO REGENERATIVE MEDICINE:-

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Figure 6:-Cell therapy

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5.4 Cell therapy challenges:-

5.5 TISSUE ENGINEERING APPROACH:-

Tissue Engineering Process

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Figure 7:-Scaffold cell delivery

Figure 8:-Process of scaffold seeding

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6.1 Scaffolds for Tissue Engineering and Regenerative medicine:-

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Figure 9:-Microscopic appearance of scaffold cell

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Figure 8:-Demonstration of use of scaffold cell

6.1 Bio-material used in scaffold:-

Material Relevant features and application

Synthetic Poly(glycolic acid) (PGA) Musculoskeletal tissue

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6.3 Roles of a Scaffold in Tissue engineering and Regenerative medicine [28]:-

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7. Application of Tissue Engineering and Regenerative Medicine

Figure 9:-Integra wound dressing

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7.2 Hair Stimulating Complex:-(HSC)[34]

Patients treated with Histogen's Hair Stimulating Complex (HSC)