Tissue Engineering
Cell Sources for Cartilage Tissue
Engineering – Ch4
Lipid-Mediated Gene Transfer for
Cartilage Tissue Engineering – Ch5
Tissue Engineering of Articular
Cartilage – Ch7
Reference: Culture of Cells for Tissue Engineering (Culture of Specialized
Cells), Chapter 4, 5, and 7
Shu-Ping Lin, Ph.D.
Date: 04.18.2011
Institute of Biomedical Engineering
E-mail: [email protected]
Website: http://web.nchu.edu.tw/pweb/users/splin/
Cartilage - 1




Many sites of permanent cartilage within the body, specific and
distinct functions depending on its location, no two cartilages are the
same  specific extracellular matrix that is produced by cells
termed chondrocytes, which are defined by their production of
type II collagen, the major collagen of most cartilage.
There are differences between chondrocytes, both within and among
different cartilaginous tissues.
All cartilage extracellular matrices have common constituent
molecules, but they are present in different proportions, with some
molecules unique to certain types of cartilage.  The specific type of
cartilage one is seeking to repair or regenerate.  What form of
initial cartilage implant is acceptable, given that the implant may
remodel in vivo into the desired cartilage type.
Chondrocytes and cells with chondrogenic differentiation
potential from embryonic and postnatal sources are currently
being used for cartilaginous tissue repair and regeneration studies.
Cartilage - 2




Network of fibers in rubbery ground substance
Resilient and can endure more stress than
loose or dense connective tissue
Differs with site, age, and species  many
cartilage types have poor intrinsic
regenerative capabilities after injury.
Types of cartilage



Hyaline cartilage
Elastic cartilage
Fibrocartilage
Hyaline Cartilage




Bluish-shiny white rubbery substance
Chondrocytes sit in spaces called lacunae
No blood vessels or nerves so repair is very slow
Reduces friction at joints as articular cartilage
Elastic Cartilage


Elastic fibers help maintain shape after
deformations
Ear, nose, vocal cartilages
5
Fibrocartilage


Many more collagen fibers causes rigidity &
stiffness
Strongest type of cartilage (intervertebral
discs)
Growth & Repair of Cartilage


Grows and repairs slowly because is
avascular
Interstitial growth



Chondrocytes divide and form new matrix
Occurs in childhood and adolescence
Appositional growth


Chondroblasts secrete matrix onto surface
Produces increase in width
Bone (Osseous) Tissue

Spongy bone




Compact bone



Sponge-like with spaces and trabeculae
Trabeculae = struts of bone surrounded by
red bone marrow
No osteons (cellular organization)
Solid, dense bone
Basic unit of structure is osteon (haversian
system)
Protects, provides for movement, stores
8
minerals, site of blood cell
formation
Compact Bone

Osteon = lamellae (rings) of mineralized matrix




Calcium & phosphate---give it its hardness
Interwoven collagen fibers provide strength
Osteocytes in spaces (lacunae) in between lamellae
Canaliculi (tiny canals) connect cell to cell
9
PREPARATION OF MEDIA AND REAGENTS-1
PREPARATION OF MEDIA AND REAGENTS-2
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Therapeutic Focus

The expertise of basic and clinical medicine, biomaterials, and engineering
to provide an improved product with the following properties:




is conveniently injectable and flows to conform to the treated surface
can strongly attach to the surrounding tissue
promotes cell growth and integration with the surrounding tissue
degrades/adsorbs slowly enough to allow stable tissue regeneration
Artificial Cartilage
Transplants
The meniscal transplant - the cartilage shock
absorber in the knee joint. If for some reason that
is torn, it has to be surgically removed and a
donated cartilage disk is transplanted into the
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knee.

Articular cartilage is laminated to the ends of the bones that are inside the joints. There
are a few different ways to transplant that kind of cartilage. 1. to take plugs of bones and
cartilage from a separate area of the patient’s joint and plug in into the area that’s
missing the cartilage. That can also be done in the ankle joint. 2. A similar transplant
procedure for articular cartilage involves removing cartilage and bone from a cadaveric
donor and transplanting that into a patient’s knee. There’s minimal rejection problem
because cartilage tends to be immunoprivileged, whereby the immune system has
difficulty coming in direct contact with immunogenic cells.

The third type of transplant is analogous to patching an automobile tire; a patient’s
healthy cartilage cells are removed and sent to a laboratory that grows and duplicates
them in tissue culture. Six weeks later they return the cells to the damaged joint. The
damaged cartilage is then excised from the patient’s knee and the test tube cells, called
chondrocytes, are inserted. Placing a piece of lining of a bone and sewing it over the area
will protect the cartilage cells so they can continue to grow and bond.
ARTICULAR CHONDROCYTES FOR
CARTILAGE TISSUE ENGINEERING
Total joint arthroplasty (TJA) specimens
can generally be obtained within hours of
removal from the patient but are overtly
pathological material and are usually
acquired from older individuals.

Shorter (3–6 h) digestion, the
collagenase concentration can be
increased up to 0.40%. The FBS can be
omitted with shorter digestion times.
Note that with higher collagenase
concentrations, filtration can be difficult.
The protocol is equally relevant to collection from other sources, such as postmortem,
amputation, and organ donor specimens.

Articular Chondrocytes from
Other Species



Several animal species are routinely
used for articular cartilage and
chondrocyte research: rabbits, pigs,
goats, dogs, horses, and cattle.  are
not compromised by preexisting
pathology, delays in acquisition, or
potential biohazardous risks.
It is possible to isolate up to 1 × 108
articular chondrocytes from extensive
collections from equine or bovine
limbs.
The protocol is equally applicable to
other experimental species, such as
dog, horse, goat, and rabbit.

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The technology provides a
convenient and effective
photopolymerization of a
hydrogel that contains
cells and growth factors
necessary for the growth
and integration of new
cartilage in a damaged
tissue
Articular Cartilage Injury: A
Permanent Injury




Poor vascularity…No healing
potential
Adult chondrocytes don’t
migrate or replicate to fill
defects
Injury begins an inexorable
cascade of events both
chemical and then
mechanical leading toward
degenerative joint disease.
May progress to end stage
arthritis
By Ken Zaslav MD, Virginia Commonwealth University
Symptoms of Articular
Cartilage Injury:
Pain
 Catching / Clicking / Locking
 Instability
 Effusions

By Ken Zaslav MD, Virginia Commonwealth University
Prevalence of Cartilage Injuries



Cartilage injuries occur frequently.1,2
 Studies suggest that 20%-60% of knee arthroscopies reveal
focal chondral or osteochondral defects.1,2
 Almost 10% of all arthroscopies in patients <50 years old,
revealed a single, well-defined grade III or IV defect with an
area of at least 1 cm2. 2
Can cause significant disability in relatively young patients.
2
 Can be painful and debilitating.
 Limits employment, sports participation, and activities of
daily living.2
Cartilage is avascular, aneural, and will not regenerate on its
own.
1.
2.
Curl et al. Cartilage Injuries: A review of 31,516 Knee Arthroscopies. J Arthrosc. Rel. Surg. 1997;13:456-60
Hjelle et al. Articular Cartilage Defects in 1,000 Knee Arthroscopies. J Arthrosc. Rel. Surg. 2002;32:730-73. 4.
By Ken Zaslav MD, Virginia Commonwealth University
Unique building block of articular
cartilage matrix is Type II collagen

Middle architectural zone called “the netting” is made
of aggregates of proteoglycans called glycosaminoglycans (GAG’s): This netting holds water i.e.: gives
this zone its hydrophilic character that yields the low
friction, fluid wave enabling smooth joint motion
By Ken Zaslav MD, Virginia Commonwealth University
Goals of Cartilage Repair




Restore smooth articular
cartilage surface
Relieve patient symptoms
and improve function
Match
biomechanical/biochemical
properties of normal hyaline
cartilage
Prevent or slow progression
of focal chondral injury to
end- stage arthritis
By Ken Zaslav MD, Virginia Commonwealth University
Early Treatment Options:




Most involved debridement to remove mechanical
symptoms (palliative only/ No Repair tissue)
Moved to marrow stimulation techniques to bring in
pluri-potential cells from the sub-chondral marrow to
fill defects. ( abrasion/ drilling/ burr)
These all yield a fibro-cartilage repair and may affect
the integrity of the sub-chondral plate: Therefore
repair tissue is not as durable as normal hyaline
articular cartilage
Late term clinical problems seen in larger lesions
By Ken Zaslav MD, Virginia Commonwealth University
Treatment Options for the
Cartilage Bio-surgeon in 2009
C
L
I
N
I
C
A
L
Debridement
& Lavage
Microfracture
Autologous
Chondrocyte Osteochondral
Grafting
Implantation
U
T
I
L
I
T
Y
Palliative
Reparative
By Ken Zaslav MD, Virginia Commonwealth University
Restorative
Treatment Decision Lesion
Algorithm
≥ 2 cm
Lesion < 2 cm2
Primary
Treatment
D&L
 MST
 Osteochondral
Autograft
2

Secondary
Treatment
 ACI
 Osteochondral
Autograft
Primary
Treatment
Low
Demand
D&L
 MST
 Osteochondral
Grafting
High
Demand
 Osteochondral
alloGrafting
Special Issues exist for
the competitive Athlete?
It is always about time and timing!
By Ken Zaslav MD, Virginia Commonwealth University
Secondary
Treatment
 ACI
 Osteochondral
alloGrafting
The Knee Joint is an organ :
Cartilage is only one component: The
organ also includes bone, soft tissue,
synovial fluid.
Co-morbidities must be corrected prior
to or concurrent with any cartilage
repair procedure:



Ligamentous stability
Mechanical alignment
Functional meniscus
Courtesy of
Brian J. Cole, MD
By Ken Zaslav MD, Virginia Commonwealth University
Microfracture


Strengths:
 Arthroscopic procedure is
relatively simple/reproducible
 Inexpensive
 Long history of clinical use
(> 28 studies w/ 6 RCT’s in lit.)
Limitations:
 Creates fibrocartilage/
poor wear characteristics
 More effective on smaller
defect (< 4 cm2)
 6–8 weeks protected- wt.
bearing and CPM required
Courtesy of Brian J. Cole, MD
By Ken Zaslav MD, Virginia Commonwealth University
1. St
2. Knee. A Randomized Trial. J of Bone Joint Surg. 2004;86-A:455-464.
Microfracture has been a good step ….but
not ideal: not truly restorative





good 2 yr clinical effect with waning
clinical effect in larger lesions
"osteochondral" perforating the
subchondral bone plate/tidemark
moving up of the bone front leading to
intralesional osteophytes
Over time deterioration of the repair tissue
Declination of function and athletic activity
Mithoefer: JBJS Am 2005; 87 (9) 1911-20
Buckwalter, Grodzinsky: Articular cartilage and osteoarthritis: Instr. Course Lect
2005; 54: 465-80
Minas; Orthopedics 1997; 20 (6) 525-38
Kreuz: Osteoarthritis and Cartilage (2006) 14, 1119-1125
Kreuz: The Journal of Arthroscopic and Related Surgery Vol 22, No
11(November) 2006, 1180-1186
Brown : Clin Orthop Relat Res 2004; 422: 214-23
By Ken Zaslav MD, Virginia Commonwealth University
Osteochondral Autograft

Strengths:



May be performed
arthroscopically/open
Fills defect with native
cartilage
Limitations:




Limited to smaller
defects
Donor site morbidity
No lateral integration
Congruity of joint
difficult to reproduce
with multiple plugs
Courtesy of Brian J. Cole, MD
1. Levy, A.S. Osteochondral Autograft ofr the Treatment of Focal Cartilage Lesions. Operative Techniques in Orthopedics. Management of Chondral Injury: Perspectives in the Millennium. 2001;11:108-114.
2. Levy, A.S. and Meire, S.W. Osteochondral Autograft Replacement. In: Cole, B.J. and Malek, M.M. Articular Cartilage Lesions. Practical Guide to Assessment and Treatment. New York, New York: Springer, ; 2004:73-81.
By Ken Zaslav MD, Virginia Commonwealth University
Osteochondral Allograft


Strengths:
 Bone fixation
 Hyaline cartilage

Fresh Allografts have
excellent long term results
(Garrett/ Gross)
Limitations:
 Limited supply
 Disease transmission
 ( partially mitigated by
cold storage: 20 days)
 Viability of chondrocytes
 appx. 20 % Non-union
Courtesy of Brian J. Cole, MD
By Ken Zaslav MD, Virginia Commonwealth University
Cell Therapy: U.S.A. Clinical Development
and Regulatory Background
1994: Brittberg-Petersen Study Published JAMA
Autologous Chondrocyte Transplantation
1995

Genzyme Corp. manufactures and
commercializes the first cell-based therapy
in orthopaedics (approved as unregulated medical
device status)
First Articular Cartilage Transplants performed in USA
1996

FDA develops new cell therapy regulations
1997

Carticel® receives FDA approval (Accelerated Approval) under new FDA cell therapy
regulations; however Post approval studies are required
1999 – 2000

Two new post approval study designs are approved by FDA
Registry based (completed 2000) Pub. Mandelbaum B, et al: Am J Sports Med 2007;35:915-921
Prospective Cohort study: (STAR) (completed 2006) Pub. Zaslav,K Cole B. et al : Am J Sports Med 2009;37(1):42-55;

By Ken Zaslav MD, Virginia Commonwealth University
Autologous Chondrocyte
Implantation : ACI

Strengths:



Can produce hyaline-like cartilage
Not limited by defect size
Most commonly used for moderateto-large defects in patients who
have failed previous interventions



15 year hx of clinical use
> 80 citations in literature
Courtesy of Jack Farr, MD
Limitations:





Open/More invasive
Expensive
Longer recovery period
2 stage procedure
Ultrastructurally still not true
articular cartilage
1. .
By Ken Zaslav MD, Virginia Commonwealth University
ACI is a 2 stage procedure:


Biopsy Procurement:
Arthroscopic harvest from
non- weight bearing,
non-articulating surface
(Best: inter-condylar notch)
2nd stage is an open surgical
implantation of cells under a
periosteal patch sewn in with 6-0
suture.
By Ken Zaslav MD, Virginia Commonwealth University
Autologous Chondrocytes - Objective
Analysis Indicates Durable Result
Second look with matching histology
Polarized
Alcian Blue Van Giesson
Patient #6
Arthroscopic Assessment = 10
Indentation
Normal 3.8-Repair Tissue 3.7
8 years post implant
By Ken Zaslav MD, Virginia Commonwealth University
Courtesy of Lars Peterson
Study of the Treatment of Articular
Repair Clinical Trial: An overview

Objective:



To examine the safety and efficacy of ACI in patients who had an
inadequate response to a prior surgical treatment for articular cartilage
defects of the knee. (FDA approved indication )
A prospective, four-year, open label, multi-center GCP -FDA
approved study of 154 patients treated with ACI.
Level II evidence: cohort study (20 Centers N. America)
 Patients had at least one symptomatic grade III or grade IV defect
(Outerbridge) located on the femoral condyle and a Modified
Cinncinnatt Score of less than 5. (First and only cartilage study to
delineate a pre-treatment severity level) .
 A challenging patient pop. : Avg. age 34.5 / mean defect size 4.63 cm
 All patients were required to have failed at least one non-ACI surgical
repair procedure. (debridement, micro-fracture or OATS)
Mean # prior knee surgeries = 1.9

1.
An industry sponsored study
Zaslav K. Cole B. et al. A Prospective Study of Autologous Chondrocyte Implantation in Patients
Who Failed Prior Treatments for Articular Cartilage The American Journal of Sports Medicine.
2009;37(1):42-55.
By Ken Zaslav MD, Virginia Commonwealth University
Results: Adverse Events: SSPs

Subsequent Surgical Procedures (SSPs) were common following ACI
implantation.

49% of patients (n=76) underwent an SSP on the treated knee,
irrespective of relationship to ACI.
 Majority of SSPs occurred within the first 24 months post implantation.
 0-6 months: lysis of adhesions was the most frequently performed
intervention
 After 6 months, periosteal debridement for hypertrophy was the most
frequently performed intervention.


All SSP’s were arthroscopic.
SSPs were not predictive of treatment failure.
 Of the patients who required an SSP, 61% (46 out of 76 patients)
went on to have successful results.1

Zaslav K. Cole B. et al: The American Journal of Sports Medicine.
2009;37(1):42-55.
By Ken Zaslav MD, Virginia Commonwealth University
Level I Evidence: RCT
Journal of Bone and Joint Surgery Am. March 2004
By Ken Zaslav MD, Virginia Commonwealth University
Comparative Outcome 5 yr
ACI vs Microfracture








RCT: Level I Evidence
No industry sponsorship
80 Patients @ 5 years
ICRS, Lysolm, SF 36 and Tegner
Study includes all patients 2-10 cm lesions
Conclusion: Clinical Outcome shows no stat. sig.
difference between treatment groups : both 77%
@5yrs
1/3 pts showed radiographic evidence OA at 5yrs.
Pts. with higher cartilage scores at 2 yrs. had no
failures at 5 yrs.
Micro Fx results best in lesions < 4 cm w/ ACI no size
Knutsen G. et al J Bone Joint Surg Am.2007;89:2105-12
effect seen
By Ken Zaslav MD, Virginia Commonwealth University
European RCT Study powered
for histology (at 12-18m)
Industry sponsored study
By Ken Zaslav MD, Virginia Commonwealth University
Next surgical step to simplify use
of existing technology and
decrease SSP’s/SAE’s :
C-ACI




Use of collagen Patch instead of periosteum in
ACI
Type I / III Porcine Bilayer collagen membrane
Removes need to harvest periosteum
British study showed significant decrease in
SSP due to patch hypertrophy or adhesions <
10%
Gooding CR, Bartlett W, Bentley G, et al. A prospective, randomized study comparing two techniques of
autologous chondrocyte implantation for osteochondral defects in the knee: Periosteum covered versus type
I/III collagen covered. Knee 2006;13:203-210
By Ken Zaslav MD, Virginia Commonwealth University
Cartilage Repair: US
Procedural Share
Small Defects
Persistent Pain
OCG
8%
ACI
1%
Allografts
1%
MST
20%
30% Revised
at 10 months
D&L
70%
By Ken Zaslav MD, Virginia
Commonwealth University
Disease Available for
future pipeline procedures
Current US Cartilage Repair Market – Value = ~ $52/660M
Next Generation Techniques:






Scaffolds to enhance Micr0-fx marrow cell
stimulation
2nd Generation Cell Techniques
Minced Cartilage ( One stage techniques)
3rd Generation cell techniques
Concurrent Use of Growth factors/ BMP’s
Enhanced Stem cell derived:
By Ken Zaslav MD, Virginia Commonwealth University
Scaffolds

Region-specific

Conductive : several substrates
Including chitosan/ fibrinogen
Bio-replaced

Cost-effective

 May act as Micro-fx adjunct
ie: Scaffold guided regeneration
By Ken Zaslav MD, Virginia Commonwealth University
2nd Generation Cell Therapies




Autogenous cells
Seeded scaffold or liquid gel
Minimizes periosteal
related complications
Allows arthroscopic implant
By Ken Zaslav MD, Virginia Commonwealth University
nd
2
Generation Cell therapy
Enhancements Continued:

Assays for Phenotypic selection

Molecular markers to find an uber chondrocyte

Possibly yield improved chondrogenesis

Possibly more durable matrix
By Ken Zaslav MD, Virginia Commonwealth University
Next Generation Techniques:






Scaffolds to enhance Micr0-fx marrow cell
stimulation
2nd Generation Cell Techniques
Minced Cartilage ( One stage techniques)
3rd Generation cell techniques
Concurrent Use of Growth factors/ BMP’s
Enhanced Stem cell derived:
By Ken Zaslav MD, Virginia Commonwealth University
Minced Cartilage





Autogenous
Allogeneic
Time =O decision making
May use scaffold/ staple
May implant in fibrin glue
Fragments +
Scaffold
By Ken Zaslav MD, Virginia Commonwealth University
3rd Generation Cell Based




Autogenous
Allogeneic
3-D Cartilage graft
Technical ease might allow
arthroscopic insertion with
bioadhesive
By Ken Zaslav MD, Virginia Commonwealth University
Other 3rd Generation Potential
Enhancements:
–
–
•
Expanded Juvenile chondrocytes
Scaffold independent cx
Clinical: Phase I completed:
–
FDA Phase II/III IND/BLA
pending
Sheep Allograft 8 Weeks
Juvenile Cartilage
Adult Cartilage
By Ken Zaslav MD, Virginia Commonwealth University
Next Generation Techniques:






Scaffolds to enhance Micr0-fx marrow
cell stimulation
2nd Generation Cell Techniques
Minced Cartilage ( One stage
techniques)
3rd Generation cell techniques
Concurrent Use of Growth factors/
BMP’s
Enhanced Stem cell derived
By Ken Zaslav MD, Virginia Commonwealth University
Articular Cartilage Healing by
OP-1( BMP-7)
Jelic et al 2004

12 Sheep (1 year, 60 kg)
1 knee, 1 trochlear defect

Deep cartilage layer intact


Cocktail of factors: continuous delivery
(28 d) via mini-osmotic pump

Arthroscopic monitoring of repair

Sacrification at 12 months
By Ken Zaslav MD, Virginia Commonwealth University
Microfracture/BMP-7(OP-1)
Results
Kuo Rodrigo et al Osteoarthritis 2006



RabbitsTrochlear ACDs
Microfracture vs Microfracture plus
collagen Type l sponge with BMP-7
Results


Microfracture mostly fibrocartilage
Microfracture plus BMP-70% hyaline or hyaline like
repair
By Ken Zaslav MD, Virginia Commonwealth University
Other Synovial Fluid Factors:
Growth Factors:
-IGF-1, FGF, TG-Beta super family
Can we stimulate these to increase GAG
synthesis after cartilage injury

Catabolic Factors: Cytokines:
- IL-1, TNF, IL-6,7,8
Can we inhibit these to avoid matrix breakdown
after cartilage injury

By Ken Zaslav MD, Virginia Commonwealth University
Next Generation Techniques:
Scaffolds to enhance Micr0-fx marrow cell
stimulation
 2nd Generation Cell Techniques
 Minced Cartilage ( One stage techniques)
 3rd
Generation cell techniques
 Concurrent Use of Growth factors/ BMP’s
 Enhanced Autologous Stem cell derived
Techniques - Early animal work promising

By Ken Zaslav MD, Virginia Commonwealth University
Ultimate Goal:
Cells
3 Key Requirements
• Biodegradable Scaffolds to
anchor, deliver and orient cells
• Bioactive factors (Reagents) to
Scaffold
provide instructional cues to cells
• Cells: responsive to their
environment therefore milieux
shape/ O2 Tension effects should be
considered to optimize growth
By Ken Zaslav MD, Virginia Commonwealth University
Reagents
Next Gen. Cartilage Repair:
Conclusions –







Exciting developments in evolution
Now is the time for Profiling and
stratification of patients
To consider issues concerning timing of
surgery and cohort details
Scaffolds and Stem cell and cellular
optimization techniques
Effective Meniscus replacements
Delineate effective growth factors
Develop Arthroscopic delivery techniques
By Ken Zaslav MD, Virginia Commonwealth University
Ultimately What Will Yield
The Perfect Clinical Result?







An effective and available cell source
Enhanced architecturally with effective scaffold
Enhanced biologically with BMP’s
Surgery: minimally invasive or arthroscopic
Single stage
Cost effective (market driven cost)
High success rates and Low complication rate
(similar to TKA)
By Ken Zaslav MD, Virginia Commonwealth University
NORMAL CARTILAGE
In The Near Future?




A dialogue among scientists, surgeons, regulators and
industry is needed to find the best paradigm to allow
new treatments available in Europe and Asia to become
available to help U.S. patients.
ICRS is ready to help facilitate this discussion: Recent
Miami summit was very successful in delineating the
problems and resources needed to address solutions
We need to move forward responsibly, however, to
avoid poor treatment paradigms and patient
complications while providing cost effective care.
Regarding cell technology: Currently neither scientists
nor industry have a clear path to design effective studies
to bring new technology to market.
By Ken Zaslav MD, Virginia Commonwealth University
Questions To Be Considered To Move
Forward With Study Design.
•
•
•
•
•
Need to consider what is the best comparator for RCT’s. Is
micro-fx the truly best comparator for all Rx
Are alternate Level I or II study designs available in other
medical lit. as option to std. RCT when needed
Should different size lesions be treated as different cohorts
rather than considering all lesions equivalent?
Should patients entered into cohorts have some validated
score for pre-RX symptoms? Or are symptomatic and
asymptomatic lesions equivalent?
What cohort sizes are needed to see statistically
significant treatment differences clinically, structurally and
histologically.
By Ken Zaslav MD, Virginia Commonwealth University
What is our largest unmet need?
35 - 40 yr. old patient
By Ken Zaslav MD, Virginia Commonwealth University