CHAPTER 15 - Medical Applications of Bioprocess Engineering

IN THIS PRESENTATION WE WILL LEARN ABOUT THE FOLLOWING:

• What is a biological product?
• What is the application for the product?
•  Is the product currently being produced commercially?
• What is the host cell that produces the product?
• What type of bioreactor is utilized?
• What types of downstream processes are utilized?
• What analysis did the author perform on the process?

Topics
 Tissue Engineering
 Gene Therapy
 Bioreactors

TISSUE ENGINEERING

What is Tissue Engineering? 

• A practice of combining cells, a biomaterial template, and growth cues to restore,
maintain, or improve tissue and organ function.
• Developing in vitro tissues based on cells derived from donor tissue.
• Used in transplants.
• Commercial examples: skin and cartilage.
• Artificial liver outside the body is in trials. Uses hollow fiber reactor and pig liver cells.
• Under development: liver, pancreas, kidney, fat, blood vessel, bone marrow, bone,
neurotransmitter secreting constructs.

NTRODUCTION
• The term “artificial skin” was first introduced by JF Burke in 1987, and used to
designate a bilayered dermal- epidermal replacement devised by Burke and Yannas.
• Now it can be applied to several on bilayered products that have been engineered
for permanent replacement of lost human dermis and that provide either a temporary
or potentially permanent epidermis.

THE STRUCTURE AND FUNCTION OF SKIN

Two distinct layers of skin:
• epidermis - keratinocytes
• dermis - fibroblasts and collagen

• SKIN STRUCTURE
• Thermoregulation
• Microbial defense (both mechanical barrier and immune defense)
• Desiccation barrier
• Mechanical defense and wound repair
• Cosmetic appearance, pigmentation, and control of contraction.

• Epidermal injury (first degree)

• Superficial dermal injury (second degree)
• Epidermal plus near-full to full dermal injury (third degree)

Autograft (Split-thickness skin grafts)

• The best material for wound closure, when practical, is the patient’s own skin
(autograft).
• Split-thickness skin grafts (epidermis plus a thin layer of dermis) harvested from the
patient’s uninjured skin is essential for closure.

• Figure 1. Integra, the bilaminate artificial skin of Burke & Yannas, applied to a
full-thickness skin defect.
• Figure 2. Integra, 1 week after application to a full-thickness skin defect.
• Figure 3. Second-stage Integra Grafting. At 2 weeks after integra
application, the process of neodermis formation is complete; the
temporary silicone epidermal analog has been removed.
• Figure 4. Second-stage Integra Grafting. A meshed ultrathin autograft has
been applied. The epidermal cells of the autograft has been applied. The
epidermal cells of autograft proliferate and attach to the underlying
neodermis, forming a durable and confluent epithelium.

SEVERAL DISADVANTAGES OF AUTOGRAFT

• The donor site is a new wound.
• The donor site is subject to scarring and pigmentation changes.
• The dermis taken from the donor site is not replaced.
• The donor site is a potential site for microbial entry.
• The donor site cannot provide an unlimited supply of dermis.
• The limited supply of donor sites on a patient.

In order to promptly close the wound, the skin substitute had to…
• Adhere to the substrate.
• Be durable and sufficiently elastic to tolerate some deformation.
• Allow evaporative water loss at the rate typical of the stratum corneum.
• Provide a microbial barrier.
• Promote hemostasis.
• Be easy to use.
• Be readily available immediately after injury.
• Elicit a "regeneration-like" response from the wound bed without evoking an
inflammatory, foreign-body, or non-self immunologic reaction.

ARTIFICIAL SKIN TISSUE REGENERATION MATRIX

• The two artificial skins that currently exist :
• Integra, devised by Burke & Yannas, was designed by applying materials science
and engineering principles to the problem of dermal replacement.
• Bell’s product, which is being commercially named Apligraf was designed by
applying the principles of tissue culture.

• First, it has no intrinsic immunologic defenses and must be kept free of bacteria.
• Second, the silicone epidermal analog is purely prosthetic and must be removed
and replaced with epidermalautograft.
• A third drawback is that Integra, although it is fairly strong and elastic, does not do
particularly well on those areas such as the back, the axilla, and the groin because
of shear stress.

Artificial skin as a Pre-engineered Tissue Substitute

• In contrast to the materials science and engineering approach
• of Burke & Yannas, Bell and colleagues took theapproach of reconstituting dermal
injury by applying a preformedtissue.
• The resulting product is described as a dermal equivalent, which, unlike Integra,
relies on living cells in tissue culture to organize the collagen network.

The drawback of Apligraf

• In order to provide definitive wound closure, an Apligraf-like product would have to
be constructed from a patient’s own fibroblasts and keratinocytes.
• The production of a patient-specific product (i.e. with fibroblasts and keratinocytes
taken from the patient) would take several weeks, during which the wound would
have to be covered with a temporary skin substitute.

Temporary Dermal Replacement

Available new products:
• Human cadaveric allograft
• Biobrane
• Dermagraf-TC or Transcyte

Deramagraft-TC
• Deramagraft-TC is a two-layer synthetic material designed as a temporary skin
substitute. The outer layer is a silicone polymer, and the inner layer is a nylon mesh.
• Scanning electron micrograph of human dermal fibroblasts grown on a three-
dimensional nylon scaffold (Dermagraft-TC).

Cartilage Engineering
Introduction
• Most people “Achilles heel” is not their achilles heel but their knees.
• The knee is not that simple, it is actually an interwoven system of ligaments,
cartilage, and muscle.

Functions of the Components

• Anterior Cruciate Ligament (ACL) : responsible for stabilizing and preventing
excessive extension and lateral movements in the joint.
• Posterior Cruciate Ligament (PCL) : responsible for stabilizing and preventing
excessive flexion and lateral movements of the joint.
• Medial Collateral Ligament (MCL) : provides stability against pressure applied to the
leg that tries to bend the lower leg sideways at the knee, away from the other leg.
• Lateral Collateral Ligament (LCL) : provides stability against pressure applied to the
leg that tries to bend the lower leg sideways at the knee, toward the other leg.
• Patellar Tendon : connects the knee cap to the tibia.
• Meniscus (Lateral and Medial) : rest on the top of the tibia and provide a shock
absorbing effect.
• Articular Cartilage : Creates a low friction surface for the joint to glide on.
Figure 1: The knee in flexion (bent)

Why does Articular Cartilage need to be Engineered?

• Replacement of the articular cartilage is a necessity because “defects in mature
articular cartilage do not heal without residues” (Reiss, Rudert, Schulze, and Wirth
141).
• Meaning that the smooth surface that the joint normally glides across becomes
rough in that area. This roughness leads to swelling, pain, and arthritis in the joint.

History of the Tissue Engineering of Cartilage Cells

• In the early 1980’s the Hospital for Joint Diseases in New York started to develop a
procedure to use the patients own articular cartilage cells to use as a transplant into the
degeneration or defect in the articular cartilage. This was do to the poor results yielded
by methods to repair the articular cartilage at that time.
• Starting in 1987 the University of Goteborg and Sahlgrenska University Hospital in
Goteborg, Sweden worked to continue the development of the new procedure.
• October of 1994 the Swedish researchers published a study in the New England
Journal of Medicine. “The Swedish researchers reported "good-to-excellent results" in
14 of 16 patients with a cartilage defect on the thigh-bone part of the knee treated at
least two years earlier. The researchers said the vast majority of patients treated on the
thigh-bone part of the knee had developed hyaline-like cartilage, similar to normal
cartilage, where the defects had been” (Genzyme “The Carticel Treatment Alternative”).
• The Harvard Health Letter rated this new technique as one of the "Top Ten Medical
Advances of1994".

The Swedish Method

• The procedure is used on patients who suffer from defects in the articular cartilage
on the bottom of the femur.
• Articular cartilage (chondral) defect before removing damaged articular cartilage.

If the defect the same type of defect as shown in Figure 2, the an Orthopedic
Surgeon will perform an arthroscopic surgery, shown in figure 3, to collect the sample
cartilage cells.
Arthroscopic surgery is a procedure where the surgeon makes three small incisions in
the knee and works with specialized equipment in a relatively noninvasive procedure.
In this new surgery first the damaged area of the articular cartilage is cut out
leaving.
Articular cartilage (surface) defect (circled in red) after removing damaged articular
cartilage.

• When the defected articular cartilage is gone the surgeon will lance off a small
amount of Periosteum, a tissue that covers the bone, taken from the medial tibia.
• The Periosteum is stitched over the hole where the defect was.
• The surgeon will then inject the new cells under the flap.
• Under the flap the cells do some additional growing and eventually connect to the
surrounding tissues to form the new cartilage.
• After the surgery each patient receives a post-operation schedule that is based on
progressive program of weight-bearing, range of motion, and muscle strengthening
exercises. Articular cartilage (surface) defect after periosteum patch is sewn in place.

Currently
• Most of the information collect has not been updated since 1999 so it is hard to
estimate the current number of surgeons that have been trained in this procedure and
just how many patients have underwent the operation.
• However, as of March 31, 1998, 2,238 surgeons had been trained in the procedure
and a total of 1,271 patients had been treated since Genzyme Tissue Repair began
marketing the product in 1995.
• In 1999, the cost of the procedure ranged from $17,000 to $38,000, with an average
cost of approximately $26,000 per procedure.
• Genzyme Tissue Repair charged $10,000 per procedure for the cells.
• The Orthopedic Surgeons in Sweden who have been using this procedure since its
conception in 1987 have recorded anywhere from a 88% to almost a 100%, depending
on the type of defect started with, improvement in the patients who they preformed this
procedure on.
 Future
• The research into articular cartilage replacement has just about run its course with no
major breakthroughs in the last five to ten years.
• However, with the number of Orthopedic Surgeons being trained in this procedure
increasing yearly the cost of the procedure should decrease while the relative safety will
increase.
• As for tissue engineering in general, there are still some problems that need to be
worked through before the engineering of complex organs can begin.
• The first issue is the complexity of the organ to be engineered. Skin and articular
cartilage are both geometrically simple organs and thus getting the cells to line up in
those formations are easy. To get the cells to line up properly and form a liver for
example takes a degree of cell control not yet mastered.
• Another issue being faced is the low blood flow through the organs. When these
organs are being grown in the laboratory the blood supply to the organs is not yet
sufficient enough for the inner cells of the thicker organs to survive.

Conclusions
• While, the Swedish method of cartilage replacement is a great innovation in the history
of mankind, there are still steps in to be taken in the cartilage replacement of the knee.
• Also, there are still many organs needed by people every year for millions of
transplants and replacements. Until viable methods to synthesize these organs are
developed the world of tissue engineering is only just beginning.

References
• 1995 Annual Report of the Whitaker Foundation: Tissue Engineering. 1995. The
Whitaker Foundation. 8, April 2002.
<http://www.whitaker.org/95_annual_report/tissue95.html>. • Burmester, G.R., M.
Sittinger, C. Perka, O. Schultz, and T. Haupl. “Joint Cartilage Regeneration by Tissue
Engineering.” Z Rheumatol 58.3 (1999): 130-5. • Genzyme Tissue Repair. 5, June 1999.
The Center for Orthopedics & Sports Medicine. 9, April 2002.
<http://www.arthroscopy.com/sp08001.html>. • Kloth, S., W. W. Minuth, and M. Sittinger.
“Tissue Engineering: Generation of Differential Artificial Tissues for Biomedical
Applications.” Cell Tissue Research 291.1 (1998): 1-11. • Reiss, G., M. Rudert, M.
Schulze, and C. J. Wirth. “Synthesis of Articular Cartilage-like Tissue In Vitro.” Arch
Orthopedic Trauma Surgery 117.3 (1998): 141-6. • Yacobucci, The Gerald N. Yacobucci,
M.D. Arthroscopic Surgery and Sports Medicine Home Page. 1999. YacoSportsMed. 8,
April 2002. <http://members.tripod.com/GeraldY/index.html>.
https://www.slideserve.com/adamdaniel/biochemical-engineering-cen-551

Using Viral Vectors

Gene Therapy
• Transfer of genes into cells for a therapeutic effect.
• Patient has faulty gene that does not encode for a correctly functioning protein.
• Genes can be delivered ex vivo (outside the body) or in vivo (inside the body).
• If ex vivo, the organ is removed, then transplanted back in.
• Genes are delivered to the cells with a virus.
• Clinic trials have been problematic.

• A normal gene may be inserted into a nonspecific location within the genome to

replace a nonfunctional gene. This approach is most common.
• An abnormal gene could be swapped for a normal gene through homologous
recombination.
• The abnormal gene could be repaired through selective reverse mutation, which
returns the gene to its normal function.
• The regulation (the degree to which a gene is turned on or off) of a particular gene
could be altered.

Viruses used in Gene Therapy

• Retroviruses - A class of viruses that can create double-stranded DNA copies of their
RNA genomes. These copies of its genome can be integrated into the chromosomes of
host cells. Human immunodeficiency virus (HIV) is a retrovirus.
• Adenoviruses - A class of viruses with double-stranded DNA genomes that cause
respiratory, intestinal, and eye infections in humans. The virus that causes the common
cold is an adenovirus.

Current Status
• FDA has not yet approved any human gene therapy product for sale.
• Current gene therapy is experimental and has not proven very successful in clinical
trials.
• In 1999, gene therapy suffered a major setback with the death of 18-year-old Jesse
Gelsinger. Jesse was participating in a gene therapy trial for ornithine transcarboxylase
deficiency (OTCD). He died from multiple organ failures 4 days after starting the
treatment. His death is believed to have been triggered by a severe immune response
to the adenovirus carrier.

Immune Response
Anytime a foreign object is introduced into human tissues, the immune system is
designed to attack the invader. The risk of stimulating the immune system in a way that
reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune
system's enhanced response to invaders it has seen before makes it difficult for gene
therapy to be repeated in patients.

Problems with Viral Vectors-Viruses

• While the carrier of choice in most gene therapy studies, present a variety of
potential problems to the patient --toxicity, immune and inflammatory responses, and
gene control and targeting issues. In addition, there is always the fear that the viral
vector, once inside the patient, may recover its ability to cause disease.
Multigene Disorders
• Conditions and disorders that arise from mutations in a single gene are the best
candidates for gene therapy. Unfortunately, some the most commonly occurring
disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis,
and diabetes, are caused by the combined effects of variations in many genes.
Multigene or multifactorial disorders such as these would be especially difficult to
treat effectively using gene therapy.

The Gelsinger Case

• OTCD occurs when a baby inherits a broken gene that prevents the liver from
making an enzyme needed to break down ammonia.
• University of Pennsylvania researchers packaged it in a replication-defective
adenovirus. To reach the target cells in the liver, the adenovirus was injected directly
into the hepatic artery that leads to that organ.
• At age 18, Jesse Gelsinger was in good health, but was not truly a healthy teenager.
He had a rare form of OTCD that appeared not to be linked to his parents, but the
genetic defect arose spontaneously in his body after birth.

• During his youth, he had many episodes of hospitalization including an incident just
a year before the OTCD trial in which he nearly died from a coma induced by liver
failure.
• A strict diet that allowed only a few grams of protein per day and a pile of pills
controlled his disease to the point where he appeared to be a normally active
teenager.
• Gelsinger received the experimental treatment in September 1999. Four days later,
he was dead.
• It appears that his immune system launched a raging attack on the adenovirus
carrier.

• FDA found a series of serious deficiencies in the way that the University of
Pennsylvania conducted the OTCD gene therapy trial,
• Researchers entered Gelsinger into the trial as a substitute for another volunteer
who dropped out, but Gelsinger's high ammonia levels at the time of the treatment
should have excluded him from the study.
• The university failed to immediately report that two patients had experienced serious
side effects from the gene therapy, as required in the study design, and the deaths of
monkeys given a similar treatment were never included in the informed consent
discussion.

Mass Production of Retrovirus

• Two part system: cell line and recombinant vector (virus).
• Cell line engineered to produce essential viral genes that have been deleted from the
viral genome.
• Virus incapable of causing disease – carriers of therapeutic genes.
• Retrovirus can only be used with dividing cells for integration of therapeutic genes.
• Require high titer of highly active viruses.

Two Obstacles Decay of Virus

• Decreasing temperature decreases decay rate more than decreases production rate.
Inhibition by proteoglycans
• Similar molecular weight as virus, so concentrated when virus is concentrated.

Stem Cells
• Differentiated cell has limited reproduction.
• Stem cells are undifferentiated cells capable of reproduction to a large number of
differentiated type cells.

Hematopoiesis
• Process of generating blood cells (8 major types).
• Hematopoitic stem cell  2 types of progenitor cells capable of replication and a
restricted range of differentiated progeny.
• Many different growth factors required.
• Different types of bioreactors being evaluated.

Artificial Liver
• Liver  metabolism, produces plasma proteins, detoxification.
• Liver can repair – but requires time.
• An artificial liver can provide regeneration time.
• In vitro hollow fiber reactors with human or pig liver cells have been examined.
• In vitro liver in clinical trials now.