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Biochemical Engineering CEN 551. Instructor: Dr. Christine Kelly Chapter 15: Medical Applications of Bioprocess Engineering. Schedule. Thursday, April 1: Dr. Hasenwinkel (hand out homework). Tuesday, April 6: Finish chapter 15.

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biochemical engineering cen 551

Biochemical EngineeringCEN 551

Instructor: Dr. Christine Kelly

Chapter 15: Medical Applications of Bioprocess Engineering

  • Thursday, April 1: Dr. Hasenwinkel (hand out homework).
  • Tuesday, April 6: Finish chapter 15.
  • Thursday April 8: Review for exam 3 (Chap. 12, 14 and 15 homework due).
  • Tuesday, April 13: Exam 3 - chapters 12, 14, and 15 and posters due.
  • Poster Presentations: Saturday afternoon, April 17.
  • Oral presentations: April 15, 20, 22, 27.
April 15: Mittal, Sameer, Xu, Anitescu

April 20: Meka, Chapeaux, Chang, Sayut

April 22: Pasenello, Prantil, Lu, Menon

April 27 Price, Reis

  • Each student will have to answer written questions about each presentation.
  • Be sure to include the answers to these questions in your presentation.
  • WWT, Chromatography and Validation: provide me a list of questions that you will answer in your presentation.
  • What is the 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?
  • Tissue Engineering
  • Gene Therapy
  • Bioreactors
what is tissue engineering
What is Tissue Engineering?

“The application of principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain or improve tissue function” (Whitaker Foundation “Tissue engineering”).

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.

The term “artificial skin” was first introduced by JF Burke in 1987, and used to designate a bilayered dermal- epidermal replacementdevised by Burke and Yannas.

Now it can be applied to several on bilayeredproducts that have been engineered for permanent replacementof lost human dermis and that provide either a temporary orpotentially permanent epidermis.

skin structure
Skin Structure
  • Skin has two distinct layers
  • epidermis
  • keratinocytes
  • dermis
  • fibroblasts and collagen
basic functions of skin
Basic functions of skin
  • Thermoregulation.
  • Microbial defense (both mechanical barrier and immune defense).
  • Desiccation barrier.
  • Mechanical defense and wound repair.
  • Cosmetic appearance, pigmentation, and control of contraction.
skin response to injury
Skin Response to Injury
  • Epidermal injury (first degree).
  • Superficial dermal injury (second degree).
  • Epidermal plus near-full to full dermal injury (third degree).
surgical management of skin loss
Surgical Management of Skin Loss

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.


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.
a few observations in designing a dermal replacement
A few observations in designing a dermal replacement
  • The thicker the dermal layer of a split-thickness skin graft, the less the graft contracts.
  • Full-thickness skin grafts contract minimally.
  • Full-thickness dermal injuries heal by contraction and hypertrophic scarring, producing subepithelial scar tissue that is nothing like the original dermis.
  • Partial-thickness wounds with superficial dermal loss heal with less hypertrophic scarring.

The two artificial skins that currently exist have sought to meet these constraints in two different ways:

  • 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.

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.

Figure 1.Integra, the bilaminate artificial skin of Burke & Yannas, applied 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 proliferate and attach to the underlying neodermis, forming a durable and confluent epithelium.


Three limitations of Intagra

  • First, it has no intrinsic immunologic defenses and must be kept freeof bacteria.
  • Second, the silicone epidermal analog ispurely prosthetic and must be removed and replaced with epidermalautograft.
  • A third drawback is that Integra, although it is fairly strongand 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 approachof 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

Inorder to provide definitive wound closure, an Apligraf-likeproduct would have to be constructed from a patient’sown fibroblasts and keratinocytes. The production of a patient-specificproduct (i.e. with fibroblasts and keratinocytes taken fromthe patient) would take several weeks, during which the woundwould have to be covered with a temporary skin substitute.



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).


Temporary Dermal Replacement

Several new products available

  • Human cadaveric allograft
  • Biobrane
  • Dermagraf-TC or Transcyte
the future of artificial skin
The Future of Artificial Skin
  • Materials science and engineering principles produced the dermalregeneration template Integra.
  • Application of tissue culture techniques producedApligraf.

In the future, a combination of materials science and tissueculture techniques is likely to produce a skin substitute thatcan function as an autograft for both dermis and epidermis.

Althoughexpensive, the new approach has demonstrated the feasibility ofcombining Integratechnology with that of tissue engineeringand may be the forerunner of 21st-century skinreplacement.


Most peoples “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
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
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
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 Swedish Method

The Swedish Method of Articular Cartilage Replacement

the swedish method1
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.

Photograph of an Arthroscopic Surgery

In the first incision the arthroscopic scope, a device that utilizes fiber optics, is inserted to allow the surgeons to see what they are doing.
  • In another incision the actual surgical cutting tool is inserted.
  • In the final incision an irrigating instrument is placed to keep the visibility of the area high.
  • Figure 4 shows the basic position of each of these tools during an arthroscopic surgery.

Arthroscopic Surgery Instrumentation

After the cells have been collected they are sent to the company Genzyme Tissue Repair in Cambridge, Massachusetts.
  • At the plant the new cells are grown, by a proprietary procedure, for a period of 2-4 weeks.
  • The cells grown are specific for the patient they were grown for.
  • After enough cells are grown they are shipped back to the Orthopedic Surgeon.
  • When the cells get back to the Orthopedic Surgeon a much more invasive open knee operation is performed.
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.

  • 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.
  • 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.
  • 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.
  • 1995 Annual Report of the Whitaker Foundation: Tissue Engineering. 1995. The Whitaker Foundation. 8, April 2002. <>.
  • 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. <>.
  • 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. <>.
gene therapy
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
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.

Adeno-associated viruses - A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19.
  • Herpes simplex viruses - A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.
current status
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.
Another major blow came in January 2003, when the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. FDA took this action after it learned that a second child treated in a French gene therapy trial had developed a leukemia-like condition. Both this child and another who had developed a similar condition in August 2002 had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID), also known as "bubble baby syndrome."
factors that have kept gene therapy from becoming an effective treatment for genetic disease
Factors that have kept gene therapy from becoming an effective treatment for genetic disease
  • Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
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 or 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
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.
models of viral infection
Models of Viral Infection

5 differential equations

  • Change in # extracellular viruses/cell.
  • Change in # internalized viruses/cell.
  • Difference = change # surface viruses/cell.
  • Change in the # endosome viruses/cell.
  • Change in the # cytoplasmic viruses/cell.
Analytical solutions can be found for the 5 virus concentrations as a function of time, each other, cell concentration, and rate constants (eqns. 15.8-12).
mass production of retrovirus
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
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
Stem Cells
  • Differentiated cell has limited reproduction.
  • Stem cells are undifferentiated cells capable of reproduction to a large number of differentiated type cells.
  • 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
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.