####### Cellular Products for Joint Surface Repair

######### March 3-4, 2005 

**Table of Contents**

[Introduction 3](#introduction)

[Meeting Goals 4](#meeting-goals)

[Regulatory Background 4](#regulatory-background)

[Joint Surface Repair---the Clinical Problem
5](#joint-surface-repairthe-clinical-problem)

[Clinical Studies 5](#clinical-studies)

[Pathophysiologic Considerations 5](#pathophysiologic-considerations)

[Cartilage Repair Options 9](#cartilage-repair-options)

[1. Debridement/lavage: 10](#debridementlavage)

[2. Stimulation Of A Repair Process From Subchondral Bone:
10](#stimulation-of-a-repair-process-from-subchondral-bone)

[a. Microfracture: 11](#a.-microfracture)

[b. Abrasion: 11](#b.-abrasion)

[c. Drilling: 11](#c.-drilling)

[1. Repair or replacement of the articular surface:
11](#repair-or-replacement-of-the-articular-surface)

[a. Mosaicplasty: 12](#a.-mosaicplasty)

[b. Tissue grafts: 12](#b.-tissue-grafts)

[c. Autologous chondrocyte implantation:
12](#c.-autologous-chondrocyte-implantation)

[Clinical Study Considerations 13](#clinical-study-considerations)

[Design: 13](#design)

[Clinical Outcomes: 14](#clinical-outcomes)

[a. Patient symptoms and knee function:
15](#a.-patient-symptoms-and-knee-function)

[b. Cartilage structure: 16](#b.-cartilage-structure)

[Clinical Questions: 17](#clinical-questions)

[Preclinical Studies 19](#preclinical-studies)

[Animal models of joint surface defects
21](#animal-models-of-joint-surface-defects)

[Small Animal Models 23](#small-animal-models)

[Large Animal Models 24](#large-animal-models)

[Canine and Porcine Models 26](#canine-and-porcine-models)

[Caprine and Ovine Models 26](#caprine-and-ovine-models)

[Equine Models 27](#equine-models)

[Immunological Considerations 28](#immunological-considerations)

[Potential Future Directions 30](#potential-future-directions)

[Preclinical Questions 30](#preclinical-questions)

[Product Characterization and Testing
32](#product-characterization-and-testing)

[Tissue source 33](#tissue-source)

[Specifications and process controls
34](#specifications-and-process-controls)

[Product Questions 37](#product-questions)

[References: 39](#references)

#   {#section .unnumbered}

# Introduction

This Cellular, Tissue and Gene Therapies Advisory Committee is assembled
to provide the Food and Drug Administration (FDA) with insight and
perspectives regarding product, preclinical and clinical concerns
confronting the development of products for the repair or replacement of
articular cartilage defects. The considerations relate specifically to
those products that contain living cells and that are administered for
correction of articular cartilage defects. The meeting discussion items
include manufacturing quality and control, preclinical considerations,
and clinical concerns related to both exploratory and confirmatory
clinical studies.

At this meeting, no specific products will be discussed or cited for
regulatory review purposes. Additionally, no data presented at the
meeting will have undergone FDA review for completeness or accuracy.
Instead, researchers in the field will present information on some of
the issues concerning development of these types of products. Members of
the committee will be requested to consider this information and provide
a response to FDA questions. While a consensus is desirable, it is not
required. Since the field is developing rapidly, FDA anticipates that
all opinions are tentative and subject to reconsideration based upon
accumulating data.

The treatment of articular cartilage defects is evolving rapidly, both
for surgical treatments and for the specific field of cartilage repair
products that contain living cells. Hence, the committee is encouraged
to anticipate the range of possible products---present and future---
that may be investigated clinically for use in cartilage repair, such as
autologous and allogeneic tissue-engineered products as well as products
consisting of cells and artificial matrices.

# 

## Meeting Goals

This meeting is arranged to accomplish the following goals regarding the
development of cellular products for the cartilage repair:

1.  To provide FDA with perspectives on the types of manufacturing and
    preclinical data critical to the initiation and completion of a
    clinical development program;

2.  To provide FDA with perspectives on the major issues in the design,
    conduct and analyses of exploratory and confirmatory clinical
    studies;

3.  To provide a public forum to discuss the major controversies in
    developing these products.

## Regulatory Background

To initiate clinical studies of investigational cartilage repair
products, a sponsor must obtain FDA concurrence, either through review
of an Investigational New Drug Application (IND) or a request for
Investigational Device Exemption (IDE), depending on whether the
investigational product is classified as a biological product or device.
The FDA website (www.FDA.gov) contains information and guidance that
sponsors may use in order to comply with the IND and/or IDE
expectations. Some investigational products may be classified as
combination products composed of two or more different regulatory
entities (e.g., device-biologic or drug-biologic). Following the
collection of sufficient manufacturing, preclinical, and clinical data,
a sponsor may submit an application to FDA for the manufacture and
marketing of a product for joint surface repair. A Biologic License
Application (BLA) is submitted for a biological product and a Premarket
Approval Application (PMA) is used for a new (unique) device. Detailed
information regarding these applications and other types of marketing
applications is also available on the FDA website. Regardless of the
regulatory pathway for marketing approval (BLA or PMA), sponsors must
provide similar supportive manufacturing, preclinical, and clinical
data.

# Joint Surface Repair---the Clinical Problem

One of the major hurdles to obtaining marketing approval for an
investigational product is the requirement that the sponsor submit
clinical data persuasive of the clinical benefit of the product. The
nature and extent of clinical data to be obtained in support of a market
application is one focus for the clinical topics to be discussed at this
meeting.

# Clinical Studies

Information cited below is intended to provide a brief summary of the
most common terms, procedures and considerations related to articular
cartilage repair, especially for readers with little prior exposure to
this field. The attached references provide substantially more detail.

## Pathophysiologic Considerations

In principle, products containing living cells might be used to repair
various joints. At present, nearly all investigational activity has
focused on the knee. Accordingly, we present the following description
of this joint and its pathobiology, with the understanding that many of
the considerations to be discussed might be applicable to other joints
as information is accumulated.

The adult knee is a complex joint consisting of the femur, tibia, and
patella, as shown in Figure 1. Also shown are certain ligaments,
tendons, and meniscal tissues that serve to stabilize the knee. The ends
of the tibia and femur (the medial and lateral condyles), as well as the
underside of the patella are covered with articular cartilage. Articular
cartilage provides a smooth, resilient surface for joint motion despite
repetitive exposure to pressure loads, shocks, or other mechanical
stresses, a limited blood supply, and little or no potential to
regenerate itself effectively.

![](media/image1.png){width="4.504166666666666in"
height="3.3784722222222223in"}

Figure 1. Major Components of the Knee

Articular cartilage consists of chondrocytes and extracellular matrix.
Chondrocytes account for approximately 5% of the tissue volume and the
matrix contributes about 95%. Certain characteristics (such as gross and
microscopic appearance) of the matrix allow cartilage to be categorized
into two major categories: hyaline or fibrous cartilage. Hyaline
cartilage is distinguished histologically by the homogenous appearance
of the matrix, especially when viewed under polarized light, and the
round or oval shape of chondrocytes. In contrast, fibrous cartilage is
characterized histologically by the presence of bundles of collagen
fibers, usually arrayed in an irregular manner when viewed under
polarized light. Fibrous cartilage is a form of dense fibrous tissue and
accounts for the cartilage found in intervertebral disks, the pubic
symphysis, and tendon insertions. The detection of fibrous cartilage in
biopsies of knee cartilage is generally thought to be indicative of
injury and suboptimal healing. In general, fibrous cartilage does not
possess the load-bearing capabilities or resilience of hyaline
cartilage.

Hyaline cartilage, the normal articular cartilage of the knee, is a
complex mixture of chondrocytes, water, and various extracellular matrix
proteins, proteoglycans, and polysaccharides. Chondrocytes are the only
normal cellular component of cartilage; the tissue contains no nerve
cells or blood vessels. Chondrocytes synthesize and secrete
extracellular matrix macromolecules, including collagens, proteoglycans
and various noncollagenous proteins. Water normally collects among the
extracellular proteins and accounts for approximately 75% of the wet
weight of articular cartilage. Most of the water within cartilage is
bound loosely within the matrix and exchanges readily with water in
synovial fluid. This water exchange allows nutritional support for
chondrocytes, a process that is thought to involve both diffusion and
mechanical pumping processes associated with joint motion and cartilage
compression. This compression---the ability to be indented by a load and
to recover from the deformity---is thought to be among the most
important functions of articular hyaline cartilage.

Articular cartilage has limited ability to repair itself following
trauma. Trauma-induced cartilage defects that do not penetrate the
entire thickness of cartilage (i.e., the subchondral bone plate) usually
remain as defects and, according to some reports, do not progress in the
absence of additional trauma. In these situations, the remaining
chondrocytes appear incapable of replication or generation of sufficient
matrix to fill the defects. In traumatic injury that penetrates the
subchondral plate, partial healing may result from fibroblastic
proliferation into the defect, a process that commonly results in the
formation of fibrous cartilage.

![](media/image2.jpeg){width="5.997222222222222in"
height="3.7395833333333335in"}

Figure 2. Histological Architecture of the Joint Surface

The clinical symptoms that result from cartilage defects are variable
and range from no discomfort to chronic pain with joint effusions and
reduced mobility, depending on factors such as the size and depth of the
lesion and patient activity level. Painful synovitis may develop in the
knee following the release of enzymatic metabolites from the damaged
cartilage. This process may culminate in arthritis, further destruction
of the cartilage, and the ultimate need for knee replacement.

Chondral defects of the knee are thought to be common. One author
reported finding a chondral lesion during 63% of over 31,000
arthroscopic procedures \[1\]. In a published arthroscopic case series
of subjects undergoing ligament surgery, the authors found that the
clinical outcomes for patients did not differ between patients who had
incidental cartilage defects and those with no defects \[2\]. This
finding supports a general understanding that asymptomatic cartilage
defects need not be treated. Nevertheless, the authors of other
published reports have expressed the opinion that some cartilage defects
will, with time, progress and culminate in immobility \[3\]. The
discrepancy between these two points of view underscores the need for
controlled clinical studies in evaluating products used to repair
cartilage defects.

## Cartilage Repair Options

The chief goal in the treatment of symptomatic cartilage defects is the
relief of pain and restoration of function \[4\]. Current treatments for
symptomatic cartilage defects include conservative management with
analgesics and anti-inflammatory agents, various rehabilitation
programs, and/or surgical intervention. Surgical treatments are
developed and modified relatively frequently. The procedures most
commonly cited in publications are summarized below. These treatments
may be divided among three categories as follows: debridement/lavage of
loose or worn articular cartilage, stimulation of a repair process from
the subchondral bone, and repair or replacement of the damaged articular
surface.

### 1. Debridement/lavage[:]{.underline}

Relatively soon after the introduction of arthroscopy into clinical
practice, lavage, a form of rinsing the knee joint, was observed to be
associated with symptomatic improvement in some patients with cartilage
defects \[5\]. The combination of lavage with debridement, (the
resection of damaged tissue) has been has been reported by some
investigators to result in better outcomes than lavage alone\[6, 7\].
Other investigators have reported the results of a controlled clinical
trial in which arthroscopic lavage or debridement produced no benefit
compared with a placebo procedure in osteoarthritis of the knee\[8\].
The pathophysiologic basis for symptomatic improvement reported
following lavage/debridement is unknown but has been attributed to the
removal of pro-inflammatory tissue. Both debridement and lavage may be
accomplished through an arthroscopic procedure.

### 2. Stimulation Of A Repair Process From Subchondral Bone:

> In these procedures, subchondral bone is penetrated surgically to
> prompt the infusion and filling of the cartilage defect with bone
> marrow and other cells that are thought to initiate repair or partial
> regeneration of the joint surface. A carefully designed post-operative
> rehabilitation program is regarded as a critical component of all
> these operative treatments. A range of clinical outcomes has been
> reported for these procedures, but few controlled, prospective
> clinical studies have been performed. In general, they result in the
> formation of predominantly fibrous cartilage, not hyaline cartilage.

#### a. Microfracture: 

> Microfracture surgery involves the use of specially designed surgical
> instruments (awls) to make multiple perforations, or microfractures,
> into the subchondral bone plate. The perforations are made as close
> together as possible within the cartilage defect but not so close that
> one perforation penetrates into another one. Preservation of the
> subchondral bone plate is regarded as an important part of the
> procedure.

#### b. Abrasion:

> Abrasion surgery involves the removal of 1 to 2 mm of the exposed bone
> within a cartilage defect using a powered burr.

#### c. Drilling:

Drilling procedures penetrate the subchondral bone within a cartilage
defect through the use of a powered drill. Although the area is infused
with saline during the procedure to cool the tissue, the heat exposure
associated with some burrs or drills has been proposed as a factor in
suboptimal cartilage repair.

### 1. Repair or replacement of the articular surface:

> These surgical procedures consist of various approaches to cover the
> surface of the cartilage defect with either autologous or allogeneic
> tissue. Variable clinical outcomes have been reported for these
> procedures and most of them are thought to result, with a few
> exceptions, in either a mixture of hyaline and fibrous cartilage or
> predominantly fibrous cartilage. These types of procedures are
> generally preferred for larger cartilage defects.

#### a. Mosaicplasty:

Autologous osteochondral mosaicplasty involves the excision of small
osteochondral cylinders from a \"minimally weight bearing\" portion of
the femoral condyles followed by implantation of these cylinders within
the cartilage defect. Major considerations in the use of mosaicplasty
include the size of the cartilage defect and the amount of tissue
available from the minimally weight bearing portions of the knee.

#### b. Tissue grafts:

> Allogeneic or autologous osteochondral tissue grafts may be implanted
> within a cartilage defect. Allogeneic grafts involve the use of fresh,
> viable cartilage (which survives) and dead bone (which is replaced by
> host bone). The technical difficulties of these procedures, combined
> with the infectious risks associated with allogeneic tissue, have
> limited their use.
>
> Both autologous periosteal patch and perichondral surface grafts have
> been used in some patients with cartilage defects. However, the
> clinical experience with these procedures is limited and reports of
> success of the procedures have varied considerably.

#### c. Autologous chondrocyte implantation:

> In this procedure, autologous cartilage tissue is harvested, the
> tissue dissociated in a laboratory, and isolated chondrocytes expanded
> in number. Subsequently, the expanded chondrocytes are implanted
> within the cartilage defect, with the cells held in place by a
> periosteal graft. At least two surgical procedures are needed, one for
> harvesting the cartilage and another, usually a few weeks later, for
> implantation of the expanded chondrocytes. One autologous chondrocyte
> product is currently licensed by FDA for clinical use as a treatment
> for patients who have failed a prior surgical procedure that was
> intended to correct the cartilage defect.

## Clinical Study Considerations

### Design:

During the development of a cartilage repair product, clinical studies
may be broadly divided into two categories: exploratory or confirmatory.
Exploratory clinical studies, the earliest clinical studies in product
development, examine the safety and bioactivity of the product, evaluate
dose-response effects, and thus provide information critical to the
design of subsequent confirmatory studies. Exploratory clinical studies
help generate a specific hypothesis that is then tested definitively in
the confirmatory clinical study.

> Options for the design of exploratory clinical studies are broad and
> largely dependent upon the study objectives. For example, an
> uncontrolled study may suffice to detect major safety concerns
> associated with the first administration of the product to human
> subjects. On the other hand, a controlled study design may be
> necessary to detect important pilot safety and treatment effects if
> the effects occur commonly during the natural history of the
> underlying condition. Confirmatory clinical studies must use a control
> group to assess the safety and/or efficacy of a product definitively.
> In general, the use of a concurrent (as opposed to historical) control
> is thought to be especially important for clinical studies that
> evaluate relatively subjective endpoints such as changes in knee pain
> or function. Hence, a controlled clinical study design is commonly
> cited as especially important for confirmatory clinical studies of
> products used in the repair of cartilage \[3\]. The choice of the
> control product or treatment is one of the major challenges in the
> design of these studies and a broad range of opinions have been
> expressed about the role of a placebo control, any specific surgical
> procedure as a control, and the use of subjects undergoing a variety
> of surgical procedures as a single control group.
>
> Various other considerations are important for confirmatory clinical
> studies of cartilage repair products, such as the need for
> standardization of surgical techniques, concomitant treatments, and
> rehabilitation programs. Additionally, the duration of follow-up and
> the types of follow-up evaluations are important issues for a sponsor
> to consider in the design of confirmatory clinical studies of
> cartilage repair products.
>
> The ability to perform controlled clinical studies in the evaluation
> of articular cartilage repair products has, at times, been regarded as
> difficult or impossible. Attached is a publication that illustrates
> many of the challenges associated with the conduct of a controlled
> study. This study is also notable for its use of a variety of
> potentially important outcome measures (Knutsen, et.al, 2004;
> attached).

### Clinical Outcomes:

The committee will be asked to discuss the importance of a number of
clinical study outcomes. The most commonly cited clinical outcomes
regarded as important for cartilage repair products and procedures
include measures of changes in patient symptoms, such as knee pain and
function. Surrogate endpoints evaluating gross or microscopic elements
of knee structure have also been proposed.

#### a. Patient symptoms and knee function:

Changes in patient symptoms in clinical studies may be evaluated with a
variety of methods, e.g., changes in pain medication usage, changes
detected through the use of patient and/or investigator questionnaires,
or specific types of scales, such as visual analogue scales of pain. In
practice, questionnaires are among the most commonly cited measures of
changes in symptoms and knee function.

Many questionnaires and knee-rating scales have been developed and
tested in clinical studies or clinical practice\[9-11\]. The most
commonly cited measures include:

> -the Lysholm scale
>
> -the Cincinnati Knee-Rating System
>
> -the American Academy of Orthopaedic Surgeons (AAOS) Sports
> Knee-Rating Scale
>
> -Activities of Daily Living of the Knee Outcome Survey
>
> -Short Form-36 (SF-36), or a subset, the SF-12
>
> -Western Ontario and McMaster Universities (WOMAC) Osteoarthritis
> Index
>
> -International Knee Documentation Committee (IKDC) Subjective Knee
> Evaluation
>
> -Knee Injury and Osteoarthritis Outcome Score (KOOS)

Several of these scales and questionnaires consist of multiple
components, only some of which relate directly to knee symptoms and/or
function. The clinical usefulness of each of these scales and/or
questionnaires is subject to many considerations, including the extent
of prior clinical data supporting the measure and the incremental
sensitivity of the measure in detecting important changes. Studies of
reliability, validity and responsiveness of several of the scales have
been published \[9-11\]. Additionally, the usefulness of any specific
measure may depend on specific study-design considerations, such as
blinding, evaluation time points, type of injury, method of treatment,
and subject characteristics.

Other measures of potential importance in the assessment of knee
function include joint range of motion and physical examination findings
(strength, alignment, ligament laxity, etc).

#### b. Cartilage structure:

Many published clinical studies have described, as an important outcome,
the extent of cartilage structural integrity following administration of
a cartilage repair product or the performance of a repair procedure. In
general, these assessments have consisted predominantly of
\"second-look\" arthroscopy (with or without biopsy) and various imaging
modalities.

Arthroscopic evaluation of cartilage repair products has, in many
published clinical reports, consisted of visual examination of the joint
surface and histopathologic analysis of biopsies. The cartilage surface
is readily visualized and a scoring system has been used to grade the
findings (ICRS, International Cartilage Repair Society macroscopic
score). Gross and microscopic findings consistent with hyaline cartilage
are regarded as preferable to appearance of fibrous cartilage or a
combination of fibrous and hyaline cartilage.

One of the major limitations of arthroscopic evaluation is the invasive
nature of the procedure. Histologic analysis provides direct information
regarding the nature of tissue contained in the repaired defect. On the
other hand, whether the gross or microscopic appearance of a joint
surface that has been treated with an investigational therapy gives an
accurate, clinically relevant indication of compressibility, load
bearing, or other functional characteristics is unknown. It is thus
appropriate to weigh the information to be gained from these approaches
against the potential morbidity that may be incurred.

The most commonly cited imaging studies of cartilage include standard
radiography and magnetic resonance imaging (MRI). Standard radiography
has very little utility in the assessment of cartilage defects because
of the focal nature of the defects. On the other hand, MRI provides much
greater information regarding cartilage integrity and is frequently
cited as the most useful noninvasive method of cartilage imaging \[12\].
Many technical details relate to successful MRI imaging of cartilage and
the utility of MRI imaging may vary considerably from center to center
based upon variations in the performance of the procedure.

# Clinical Questions:

1.  Confirmatory clinical studies are controlled studies designed to
    test hypotheses generated from the exploratory clinical studies.
    They should provide definitive information for licensure or
    marketing approval. The primary endpoint for a confirmatory clinical
    study should be a clear, meaningful measure of clinical benefit.
    Please discuss the extent to which each of the endpoints listed
    below meet this need. Please cite any other endpoints you regard as
    important for confirmatory clinical studies. Please note that the
    list includes some endpoints that have been regarded as clearly
    clinically meaningful measures and others that have been regarded as
    useful but not definitive measures of clinical benefit.

    a.  Changes in knee function as measured by scoring systems such as
        the WOMAC function score.

    b.  Changes in pain as measured by scoring systems that take
        medication usage into account.

    c.  Changes in clinical examination findings (e.g., range of motion,
        patient's global assessment, etc.).

    d.  Changes in the appearance of the joint surface on arthroscopy
        and histopathological appearance of a biopsy sample from the
        treated site. Please also discuss whether the potential
        morbidity entailed by biopsy outweighs its utility as an
        endpoint.

    e.  Changes in the appearance of the joint surface and joint space
        on Magnetic Resonance Imaging or other noninvasive techniques
        (e.g., X-ray, computerized tomography, etc.).

2.  Confirmatory clinical studies should provide robust, verifiable
    evidence of the clinical benefit afforded by a cartilage repair
    product. Please discuss the importance and limitations of the
    following aspects of clinical study design for sponsors to consider
    when designing confirmatory clinical studies. Please highlight those
    situations where flexibility may be acceptable and identify any
    ancillary considerations that might optimize the clinical study
    design.

    a.  The nature of the control group; for example, active product or
        active dose comparator, surgical procedure comparator,
        historical comparator, etc.

    b.  The importance of blinding procedures; for example, complete
        blinding versus the use of blinded evaluators or other options.

    c.  The duration of the clinical studies, as it relates to assessing
        short term as well as long term benefit in time weighted or
        landmark analyses. Specifically, at what time points should
        important endpoints be evaluated in order to assess the success
        and durability of a treatment effect?

# Preclinical Studies

Prior to initiation of human trials, several types of information must
be gathered in non-clinical models. Classical "small molecule" drug
development programs have focused on pharmacology and toxicology
studies, but other kinds of data are also important, especially when
relatively little is known about the entire class of experimental
products under investigation. Studies to provide a scientific and
medical rationale for evaluating the experimental product in humans, to
support an initial human dose, and to evaluate potential toxicities are
well-accepted components of most pharmaceutical development programs.
Due to their inherent complexity, products containing cells require
substantial additional information. These data may be gathered through
numerous different studies, including the following assessments: 1)
interactions between cellular and device components of a combination
product; 2) biocompatibility analysis of the device component; 3)
analysis of the contributions of different components of a product to
its biological action; 4) evaluation of potential immune responses to
the product; 5) exploration of potential clinical or surrogate
endpoints; and 6) qualification of analytical tests used during
manufacture of the product and for lot release. It is therefore
reasonable to expect that various experimental systems would be combined
to assemble a data set sufficiently comprehensive to allow sound
decisions regarding conduct of initial clinical studies. The models
selected should reflect the type of information needed for the product
in question.

One of the most basic requirements in any pharmaceutical development
program is data to provide reasonable assurance of the product's safety.
In addition to conventional safety studies that assess potential
toxicities in a context designed to model the clinical indication, many
cellular products need to be evaluated for their potential to undergo
unanticipated undesired changes in their characteristics, such as
malignant transformation. Models designed to address this concern should
evaluate a number of cells sufficient to detect rare events with
reasonable statistical confidence. It is also important to assess the
potential for adverse events in models approximating the human clinical
situation. Frequently, this information can be gained in the same
studies used to confirm the scientific and medical rationale for the
approach ("proof-of-concept" studies). These studies can also be used to
collect information on the physiological disposition of the study agent.
For combination products, routine device biocompatibility testing and
testing of the biocompatibility of the device/matrix component with the
cellular component may also be incorporated, if appropriate. Regardless
of whether a disease model or healthy animals are used, studies intended
primarily to provide safety data are termed "pivotal toxicology
studies".

Proof-of-concept studies should mimic the intended clinical indication
as closely as possible. This is needed not only to allow the most
reliable evaluation of the therapeutic potential of the experimental
product, but also to assess the likely duration of clinical effect. The
latter consideration is of special importance, because given the risks
inherent in any cellular therapy, failure of the intervention after a
brief interval of benefit could be viewed either as a late-occurring
toxicity or a treatment failure. For joint lesions, this presents
special problems, because the model needs to resemble a human patient
not only in applicable cell biology and pathophysiology, but also in
joint mechanics and anatomy.

Animal studies may also be used to evaluate specific characteristics of
an experimental product. An example relevant to the repair of joint
surfaces is the use of immunodeficient rodents to test whether a product
candidate is capable of forming stable articular cartilage *in* *vivo*.
While not addressing conventional issues of safety or proof-of-concept
per se, such experiments may provide valuable, or even crucial insight
into critical parameters for product manufacture or the suitability of
potential release tests.

## Animal models of joint surface defects

Anatomic and mechanical considerations inherent to the microenvironment
of the articular surface in which the product is implanted are important
potential determinants of the activity and safety profiles\[13-15\] of
cellular or combination products for articular cartilage repair and
regeneration. For example, preexisting instructive factors present in
the native microenvironment can influence the *in situ* differentiation
of cellular products\[13, 16, 17\]. Conversely, exogenous cells may have
the capacity to alter proliferation and differentiation of the patient's
cells. In addition, mechanical forces such as static loading from
standing and dynamic loading from locomotion greatly affect the local
microenvironment at the articular surface and have been shown to
influence the growth and differentiation potential of cultured
cells\[18-20\] and the *in vivo* durability of products intended for
cartilage repair. Various species differ markedly with respect to extent
of cellularity, extracellular matrix, and overall organization\[21\].
These additional interspecies differences in microenvironment and cell
biology may also limit inferences that can be made regarding the
eventual clinical performance of these products.

The human articular cartilage injury most frequently considered for
treatment with cellular therapy is a partial thickness injury that does
not extend to the subchondral bone. The characteristics (i.e., size,
shape, and depth of the experimental defect) in the animal model should
mirror the dimensions of the clinical defects as closely as possible.
The degree to which this is achievable depends in part on the critical
size defect (CSD), which is the smallest defect in the native cartilage
that will not heal without intervention. When injuries exceed the human
CSD, they are unlikely to heal naturally, and are therefore appropriate
targets for therapeutic intervention. Such injuries are modeled in
animals by attempting to create partial thickness lesions. This is done
by removing the superficial portion of cartilage (above the tidemark)
while preserving an intact barrier to the underlying subchondral bone
and marrow. In practice this has proven to be difficult technically.
Penetration or microfracture of the subchondral bone, either prior to or
immediately after placement of an experimental product, allows cellular
components of the native marrow to compete with the experimental product
for healing of the lesion. This frequently results in fibrocartilaginous
repair in both control (untreated) and treated lesions, which may
preclude unambiguous evaluation of the therapeutic effect of the product
itself. In fact, penetration of the subchondral bone essentially
produces a model of the surgical cartilage repair procedure known as
microfracture, as described in the previous section. Consequently, the
depth of native cartilage is a key factor in animal species selection.
The size of the CSD varies by species (Table 1), as does the thickness
of the articular cartilage. Useful disease models should thus afford
adequate cartilage thickness and an appropriate CSD.

Review of the published literature reveals that numerous joints in
several different animal species have been used to evaluate cartilage
repair products \[15, 22, 23\](Table 1). The stifle, which is the
quadruped joint resembling the human knee joint most closely, has been
the most frequently reported in the literature. Species diversity is
also reflected in differences in articular anatomy, cartilage thickness,
cartilage histology, cell biology, and age at skeletal maturity (Table
1). Within the stifle there are several articular surfaces available for
experimentation: the femoral condyle, the trochlear groove, and the
patella, which articulates with the trochlear groove and therefore has
also been used as a potential test site. The degree of loading (both
static and dynamic force) on the articular areas varies due to
species-specific intraarticular anatomy and mechanics. Therefore the
most common site of clinical damage, the femoral condyle, is not always
the appropriate site to use in the animal to mimic the intended clinical
indication. For example, in a commonly used animal species such as the
goat, a periosteal flap used to secure the cellular product in the
defect may fail due to the inherent loading forces on the femoral
condyle and therefore compromise the activity of the cellular product.
This inherent diversity among various animal models thus also
secondarily influences the choice of experimental defect site, the study
duration, and the depth and cross-sectional area of experimental
cartilage defect available for testing. For example, the area of the
articular surface available for defect creation and product implantation
is related not solely to articular size, but also to the additional
features such as condylar curvature and weight-bearing characteristics.
In the case of testing cellular products these constraints also may
limit the size of the product that can be produced.

### Small Animal Models

> The rabbit, which is the most commonly used small animal species in
> joint repair, has the advantages of lower cost and relatively early
> skeletal maturation (approximately nine months), as compared to the
> large animal species that have been used in this field (see Table
> 1)\[22, 24, 25\]. All four major articular surfaces of the stifle
> (femoral condyle, trochlear groove, tibial plateau, patella) of the
> rabbit have been reported in the literature\[26, 27\]. The relatively
> small area of the rabbit's articular surface and depth of articular
> cartilage restricts the size of the defect that can be created and
> therefore the volume (dose) of cellular implant that can be tested. In
> addition, the small size of the articular surface limits the type of
> potential device components that can be tested in the rabbit for
> fixation of cartilage repair product. For example, full size
> (clinical) versions of screws and staples cannot be tested within the
> rabbit stifle. Another concern is that the rate and degree of repair
> of cartilage damage in the adult rabbit is typically more rapid and
> more complete than that seen in adult large animals or humans. It has
> been hypothesized that this is a consequence of the rabbit's
> relatively high metabolic rate, the availability of relatively large
> numbers of pluripotent stem cells in proximity to the articular
> surface, and the relatively small volume of defect that can be
> created. Additionally the relatively light body weight of the rabbit
> decreases its utility as a model to test the durability of cartilage
> repair products. The maximal duration for a rabbit study is typically
> only six to eight weeks. Thus many interventions that appeared to show
> great promise in leporine models, as evidenced by complete
> reconstitution of normal appearing articular cartilage after fairly
> brief periods, were not useful in other systems. The rabbit has thus
> been used primarily as an *in vivo* model in the early stages of
> product development to evaluate biocompatibility, and screen various
> biomaterials for potential applicability as a scaffold/matrix, as well
> as to explore basic product design issues of the scaffold/matrix
> components of combination products.

### Large Animal Models

The dog, pig, sheep, goat, and horse have all been used as large animal
models of cartilage repair. In general, the size of the stifle increases
in proportion to the size of the animal. Therefore, the larger animals
allow for the testing of cellular products and associated attachment
devices that more closely approximate the size and design of the
intended clinical product. However larger animals also require
significantly longer to reach skeletal maturity than smaller animals.
Skeletal maturity is coincident with microenvironmental changes in the
articular cartilage that strongly influence the overall repair process.
The cartilage of all adult animals is more resistant to repair than
immature animals, and therefore more similar to adult human subjects.
Therefore therapies intended to treat injuries in adult humans should be
evaluated in animals that have reached skeletal maturity.

> The combination of overall increased stifle size, less effective
> native cartilage repair, and longer lifespan are advantages of large
> animal models of human clinical indications. The two primary benefits
> of large animals are the ability to model a clinically useful duration
> of response (durability) to products more closely and the potential to
> incorporate minimally invasive or non-invasive endpoints into a
> product development strategy prior to clinical trials. As mentioned
> previously, one key requirement for successful implementation of
> cell-based therapies for joint surface repair is durability of
> clinical benefit. Due to the biology of cartilage repair in large
> animals, studies of eight to twelve weeks duration (maximal length in
> rabbits) are only adequate to provide information on the
> biocompatibility and early cellular viability in larger animals.
> Longer-term studies of at least six to twelve months duration are
> needed to assess the true success of cartilage repair. This study
> duration is similar to what is generally thought by the orthopedic
> community to be needed for initial clinical indications of activity in
> humans. Additionally, some have attempted to model clinical
> rehabilitation regimes such as immediate post-operative rest and
> continuous passive motion can be modeled to some extent in large
> animal models of cartilage repair\[28\]. The ability to use large
> animals to test not just the cartilage repair product, but also the
> feasibility of various diagnostic modalities such as imaging,
> biomechanical tests, arthroscopy, and arthroscopic biopsy for the *in
> situ* evaluation of the product could prove to be beneficial in an
> overall product development scheme that includes these modalities in
> clinical trial design.

### Canine and Porcine Models

> Of the large animal species, dogs and pigs are used infrequently for
> the testing of cellular products for cartilage repair\[15, 29\]. The
> most commonly used surfaces in these two species are the femoral
> condyle and the trochlear groove, although the patella has been
> reported as a target site in the canine models. The pig stifle joint
> angle is unusual because it has a reduced range of motion relative to
> other quadruped models\[30\]. The intermediate size of the stifle in
> dogs and unusual anatomy in the pig suggest that these animals may be
> most effective in providing a bridge from initial product materials
> and *in situ* biocompatibility testing in rabbits to more extensive
> *in vivo* testing in sheep, goats or horses.

### Caprine and Ovine Models

Some investigators have also made extensive use of sheep (whose stifle
resembles that of goats in many respects), but by far the most
frequently used large animal in cartilage repair studies is the goat
\[16, 31-34\]. The femoral condyle and trochlear groove in the goat are
the most frequently used articular surfaces, but some investigators have
also used the tibial plateau, the articular surface that opposes and
articulates with the femoral condyle. In addition, the patella or
lateral femoral condyle of the goat has been used as a non-weight
bearing site for product implantation or harvest of source material for
autologous cellular products\[34\]. The popularity of the goat as a
model appears to be due to a combination of 1) reasonable cartilage
thickness, 2) relatively large stifle size, and 3) ease of use, cost,
and availability. Unfortunately, although the stifle is relatively
large, the anatomy of the joint is not conducive to routine arthroscopic
examination.

### Equine Models

In the horse model, the femoral lateral trochlear ridge and femoral
condyle have been used most frequently as test sites. The size of the
horse's articular surfaces and depth of the chondral plate provide an
animal model that resembles humans most closely. The equine model thus
allows for large, clinically relevant defects as well as multiple
defects per stifle\[23, 35, 36\], thereby facilitating the testing of
clinically relevant volumes/amounts/ doses of the cellular product in
defects of the size that occur in humans. Unlike the other animals
models used in cartilage repair, the size of the stifle in horses also
is sufficient to allow routine arthroscopy for interim visualization of
the joint surface and biopsy during a long-term study without
sacrificing the animal. This diagnostic modality could thus be
correlated with clinical endpoints prior to conducting a human clinical
trial\[37-39\]. In addition, the ability to conduct arthroscopy in a
horse model allows the testing of arthroscopic placement of experimental
products in cartilage lesions, which would permit direct evaluation of
this method for the delivery of products for joint surface repair prior
to use in humans.

Table 1. Animal Models used in the assessment of cartilage repair

+---------+------------+--------+--------+----------+--------+-------+
| Species | Breed      | Age at | Weight | Defect   | Car    | Cri   |
|         |            | Sk     | at     | Commonly | tilage | tical |
|         |            | eletal | Sk     | Used     | Thi    | Size  |
|         |            | Ma     | eletal |          | ckness | D     |
|         |            | turity | Ma     |          | at     | efect |
|         |            |        | turity |          | F      |       |
|         |            | (      |        |          | emoral | \     |
|         |            | years) | (kg)   |          | C      | (mm\) |
|         |            |        |        |          | ondyle |       |
|         |            |        |        |          | (mm)   |       |
+---------+------------+--------+--------+----------+--------+-------+
| Rabbit  | New        | 0.75   | 3-4    | FC, TG,  | 0.2    | 3     |
|         | Zealand    |        |        | TP, P    | 5-0.75 |       |
| (Le     | White      |        |        |          |        |       |
| porine) |            |        |        |          |        |       |
+---------+------------+--------+--------+----------+--------+-------+
| Dog     | Mixed,     | 1-2    | 15-30  | FC, TG,  | 1.3    |       |
|         | Beagle     |        |        | P        |        |       |
| (       |            |        |        |          |        |       |
| Canine) |            |        |        |          |        |       |
+---------+------------+--------+--------+----------+--------+-------+
| Pig     | Minipig    | 0.8-1  | 20-40  | FC, TG   |        |       |
|         |            |        |        |          |        |       |
| (P      |            |        |        |          |        |       |
| orcine) |            |        |        |          |        |       |
+---------+------------+--------+--------+----------+--------+-------+
| Sheep   | Suffolk,   | 2-3    | 35-80  | FC, TG   | 1.7    | 7     |
|         | Texel      |        |        |          |        |       |
| (Ovine) |            |        |        |          |        |       |
+---------+------------+--------+--------+----------+--------+-------+
| Goat    | Dairy,     | 2-3    | 40-70  | FC       | 1.5-2  |       |
|         | Boer       |        |        | ,TG,TP,P |        |       |
| (C      | Cross,     |        |        |          |        |       |
| aprine) | Spanish    |        |        |          |        |       |
+---------+------------+--------+--------+----------+--------+-------+
| Horse   | Mixed,     | 2-4    | 4      | FC, TG,  | 2-3    | 9     |
|         | Tho        |        | 00-500 | RC       |        |       |
| (       | roughbred, |        |        |          |        |       |
| Equine) | Quarter    |        |        |          |        |       |
|         | Horse      |        |        |          |        |       |
+---------+------------+--------+--------+----------+--------+-------+
| Human   | N/A        | 16-21  | 70     | FC       | 2-3    |       |
+---------+------------+--------+--------+----------+--------+-------+

FC- femoral condyle

TG trochlear grove

TP- tibial plateau

P- patella

RC- radial carpal

## Immunological Considerations

Though the articular space is thought to be an area of relative immune
privilege in both animals and humans due to the relative lack of local
microvasculature and relative hypocellularity of articular cartilage and
synovial fluid that bathes the articular surface, rheumatoid arthritis
and other inflammatory arthritides suggest that this is not absolute,
and therefore immune response to both cellular and device components of
combination products (see above) may occur in the articular space. The
testing of cellular products derived from human cells in animal models
thus poses a special concern, as these cells are xenogeneic to all
animal species, and therefore at risk for xenotransplant rejection.

Immunological reactions to human product in animals often necessitate
that preclinical studies be performed with animal cellular products that
are analogous to the intended clinical product, rather than the actual
human product. The determination that a specific animal cell is
analogous to the intended clinical product is made on the basis of some
combination of morphology, biochemical or molecular biological
characteristics, ontogeny, and function. Ideally this determination
would be multifaceted and involve not just *in vitro* measures of cell
identity, but also incorporate detailed understanding of the *in vivo*
activity of both the animal analog and putative human correlate cell.
This approach is similar to what is frequently done during preclinical
testing of monoclonal antibodies directed against epitopes expressed
only in humans. In this situation, an immune response or lack of an
applicable epitope limit the ability of the model to evaluate the
clinical product. Implicit in the use of analogous animal cells as a
means to assess biological activity and/or safety of a human cellular
clinical product that is composed at least in part of human cells is the
assumption that cells from the two species will respond similarly to the
stresses imposed in the *in vivo* articular environment. The data
obtained from testing analogous animal cells will provide a partial
basis from which to make a risk/benefit analysis that is integral to
review of preclinical data prior to initiation of clinical trials. The
degree of understanding of the relationship between an animal cell and
its human correlate is an important factor in determining the strength
of the extrapolations from findings in animals to the potential risks in
humans. The degree to which inherent interspecies biological differences
may limit inferences that can be drawn between non-human and clinical
studies is not yet known This underscores the importance of additional
work to characterize the differences and similarities between species
used for preclinical models and humans.

## Potential Future Directions

More detailed study of the comparative developmental biology of human
and important animal models is likely to uncover characteristics common
to the relevant species. For example, though details of terminal
differentiation pathways may differ, it is probable that at least some
key signal transduction pathways known to be highly conserved
phylogenetically will function analogously between species, allowing
detailed evaluation of biological responses, both *in vitro* and *in
vivo.* These data, in turn, may provide a basis for developing more
refined release tests and process controls than exist currently. *In
vivo* data from the most appropriate animal models is likely to be of
critical importance in evaluating the usefulness of such tests.

# 

# Preclinical Questions

3.  Please discuss the limitations and capabilities of available animal
    models for predicting safety and clinical activity, focusing on the
    following:

    a.  How should questions of dose and allometric scaling (i.e., size
        and shape of animal joint versus size and shape of human joint)
        be explored in animal models?

    b.  To what extent do differences between human versus animal
        anatomy and cell physiology need to be addressed in an animal
        model that uses analogous animal cells to model human
        chondrocyte function? Which specific interspecies differences
        affect the types of conclusions that can be drawn from animal
        studies?

    c.  Are non-invasive imaging modalities such as ultrasound, CT, or
        MRI adequate as a replacement for interim sacrifices in
        long-term (six to eighteen month) studies to evaluate for
        intraarticular toxicity and /or cartilage formation?

    d.  What role should biomechanical tests play in analysis of
        cartilage repair in animal models?

    e.  What role should arthroscopic biopsy play in analysis of
        cartilage repair in animal models?

    f.  Are tumorigenicity studies needed for cultured chondrocyte
        cellular products?

4.  Please provide specific comments on the following with respect to a
    "pivotal" animal toxicology study that is designed to support a
    clinical trial of a cellular cartilage repair product?

    a.  What animal model(s) and study duration are needed to support
        exploratory clinical trials?

    b.  What animal model(s) and study duration are needed to support a
        licensing application?

    c.  Traditionally, *in vivo* toxicology studies include measures of
        systemic toxicity such as clinical pathology tests and
        histopathology of major organs. Is this approach warranted for
        toxicology studies with the following categories of products:

        i.  cellular products?

        ii. modified cellular products that may secrete molecules
            capable of producing systemic toxicities (e.g., *ex vivo*
            gene therapy)?

5.  For an allogeneic cellular product for articular repair, what, if
    any, additional safety concerns beyond those posed by an autologous
    product should be addressed in an *in vivo* study prior to
    initiation of clinical trials?

# Product Characterization and Testing

During the development of a product from the exploratory investigational
phase through marketing approval, much needs to be learned about the
product and critical aspects of its manufacture. The initial focus is on
information to support a rationale for human experimentation and to
provide reasonable assurance that human subjects will not be placed at
unreasonable risk. The immediate application of this data is to allow
exploratory safety studies in humans. As development of a product
proceeds, additional experience with the manufacturing process and
ongoing characterization studies provide more detailed information about
the product itself and key parameters in its manufacture. Ultimately,
enough is learned to develop an overall strategy to ensure product
quality and consistency.

Product reliability and consistency depend primarily on three elements:
control of source materials, control of the manufacturing process, and
meaningful release tests. Somatic cell therapy products present special
challenges in each of these areas. The source material for cellular
products for cartilage repair may be tissue obtained by biopsy of the
subject or an allogeneic donor. Typically, cartilage from the joint to
be treated is used, but other tissues and anatomic sites have been
considered. Substantial inherent biological variability is thus
unavoidable. Much remains to be learned about how to manufacture cells
with clinically useful properties. Cellular products are much more
complex biochemically than other drugs or biologics, and the
capabilities of conventional analytical methods are limited. The source
material, manufacturing intermediates, and the final product may all
have very short shelf lives, imposing the further constraint that
testing methods must be rapid.

To meet this challenge, detailed studies to explore which
characteristics are important determinants of product safety or
effectiveness should begin as early as possible. As product development
proceeds, the analytical modalities that might best be used to evaluate
the product at intermediate stages of manufacture (in-process testing)
and to release the product for clinical use (specifications) can be
identified. Both types of testing play an essential role in optimizing
the entire manufacturing process and ensuring that a quality product
will be produced consistently. This level of control is needed to
support reliable conclusions about the clinical effectiveness of the
product, and is therefore essential for marketing approval.

## Tissue source

The majority of cell products intended to repair joint surfaces have
been derived from autologous articular cartilage, though recent data
suggest that other tissue sources (e.g., periosteum, synovium) might
also be considered \[40, 41\]. The tissue chosen for preparation of the
cellular product is perhaps the most critical of the starting materials;
it is thus important that acceptance criteria for such tissue be
established. Some criteria may be apparent at the time of the biopsy
procedure. For example, anatomic site, pathologic involvement of the
tissue, and overall condition of the patient---systemic disease, age,
etc.---may all affect the quality of the final product. In addition,
other characteristics of the tissue measurable by laboratory procedures
(e.g., histological analysis, biochemical, measures of gene expression,
immunassays, etc.) at the start of manufacture might be useful in
determining whether a particular tissue sample will be acceptable for
production of a safe and efficacious cellular product.

## Specifications and process controls

Reliable approaches to characterization of conventional 'small molecule'
drugs are now well understood, and contemporary methods are sufficient
for most protein therapeutics. In contrast, analytical techniques
currently available for characterizing products with the complexity of
living cells or cell populations fall far short of meeting standards
expected for the former two classes of products.

Early approaches to characterization of cellular products were often
limited to visual assessment of morphology, cell count, and assessment
of viability based on exclusion of a vital dye. These methods are
inadequate to identify many cell types unambiguously, do not accurately
reflect the capability of cellular products to adopt desirable
characteristics *in vivo*, and are very insensitive to impurities in the
cell population or to significant impairment of cellular metabolism (see
below). More recent approaches have included evaluation of cell surface
antigens by immunofluorescence microscopy or flow cytometry, assessment
of characteristic mRNAs using RT-PCR or microarray, various biochemical
analyses, and release of specific bioactive molecules or other
functional assays. To date, no publications have appeared that describe
use of these approaches to identify characteristics that predict
reliable performance of cellular therapies in joint repair.

This problem is compounded for cells used for repair of joint surfaces,
because the characteristics they adopt during culture are often very
different from those of normal articular cartilage. Thus, the customary
focus on characteristics---morphological, molecular, or
functional---commonly associated with the desired terminally
differentiated tissue has limited utility. Instead, exploratory studies
may be necessary to identify other product characteristics, some of
which may not yet be known, that are maintained or developed during
product manufacture and are sufficient to ensure the desired clinical
benefit. Conversely, the conditions used to culture and expand many of
these products are so different from the *in vivo* situation that they
may promote the appearance of undesired cell types (e.g.,
inappropriately differentiated or transformed cells) or other product
properties (extracellular matrix components or cell surface antigens
associated with pathologic states, etc.). Sensitive assays to detect
such potential impurities will therefore also be important.

It is generally assumed that for cultured chondrocytes to be effective
in cartilage repair they should express type II collagen and aggrecan
but not type I or type X collagen. Type II collagen and aggrecan
proteoglycan are the major matrix components of articular cartilage.
Conversely, type I and X collagens are not normally expressed by
articular chondrocytes and are indicative of de-differentiation and
terminal differentiation, respectively. However, it is by no means clear
that evaluation of the final product for these markers suffices to
provide assurance of satisfactory product performance *in vivo*. It is
likely that more comprehensive studies will be required to identify sets
of characteristics that can be used to identify cells that will form
clinically useful articular cartilage*.*

Combination products, or products in which cells have organized into
structures resembling formed tissues, may require special types of
studies. Many approaches already in use for evaluation of conventional
medical devices can be applied without modification to artificial matrix
components. When cells are embedded in a natural or artificial matrix,
it may be necessary to sample portions of a product for testing that may
be destructive. In some cases, destructive and nondestructive test
methods could be compared during characterization studies to determine
whether the nondestructive test is sufficient during routine
manufacturing operations. Whether any forms of mechanical testing for
certain products may be appropriate remains to be determined. Another
possible complexity for such products is that their properties may vary
across three-dimensional space. Thus, differences in spatial
distribution (e.g., in gene expression, histology, immunohistochemical
markers, etc.) may need to be assessed.

In addition to tests that will evaluate identity and purity, it is
important to assess the biological activity, or potency, of the product.
The ideal potency assay should measure a relevant biological activity of
the product quantitatively. Suitable assays for potency are often
particularly challenging to develop. For example, methods based
primarily on cell count and exclusion of vital dye give no assurance
that a cellular product will perform as intended *in vivo*. This
approach does not address any characteristic of the product related
specifically to its ability to reconstitute a joint surface. Moreover,
dye exclusion is often insensitive to substantial disturbances in cell
function and may not be applicable to products in which cells are
surrounded by naturally or artificial matrix impermeable to most dyes.
For other types of products, alternative assays, including oxygen uptake
and ATP content (Hering, B.J. and Pappas, C., *personal communication*)
show significant promise. Dyes sensitive to various aspects of cellular
metabolism, or more detailed analyses of high energy phosphoryl
compounds by mass spectrometry \[42\] or ^31^P NMR \[43\] might also be
applicable to cellular products for joint repair.

Historically, animal studies have been used to assess potency in cases
where *in vitro* assays were problematic. As noted previously, the short
shelf life of cellular products for cartilage repair may limit the
practicality of this approach. A useful compromise would be a set of
tests that would be feasible in a manufacturing environment that could
be qualified by using performance in one or more animal models as a
'gold standard'.

In summary, some product characteristics that can both be measured prior
to administration and also predict beneficial or harmful behavior *in
vivo* may be known; others may not be apparent *a priori*.
Characterization studies to address this question are therefore of
special importance. These studies might be facilitated by, or even
require, additional insights into the biology of cartilage specification
and determination in humans and model organisms together with the
application of novel analytical techniques, some of which might not be
practical in a manufacturing environment, but could help identify
suitable methods.

One requirement common to all such studies is a means to evaluate the
influence of various product characteristics on performance *in vivo*.
One of the most promising approaches is to relate product
characteristics to performance in one or more preclinical models, as
discussed above for potency. Whether or not this will prove practical
depends on the capabilities and limitations of these models, as
discussed earlier. Careful work to explore the relationship between
analytical tests suitable for use in a manufacturing environment and
performance of experimental products in various model animals and humans
is thus likely to be an important step on the critical path toward
development of cellular products for joint repair.

# Product Questions

6.  Characteristics of the starting tissue, which could be derived from
    the involved joint or a different site, may influence the quality of
    the cellular product substantially. Please discuss what criteria
    should be used for obtaining such tissue (e.g., anatomic site,
    pathologic involvement of the tissue to be collected, etc.). Please
    discuss the gross characteristics that would be useful, including
    those that may be visible at operation before excising the biopsy,
    and/or microscopic or molecular characteristics evaluated following
    collection but before use.

7.  Noting that cells intended for repair of joint surfaces, when grown
    *in vitro,* may not express characteristics of cells that produce
    differentiated cartilage, please consider the following question:
    What characteristics (e.g., based on analysis of proteins,
    extracellular matrix components, mRNA expression levels, cell
    surface antigens, cellular morphology, functional properties, or
    other parameters) could be used to identify cells that will form
    stable chondrocytes *in vivo*? Where appropriate, discuss
    characteristics that should be absent from these products as well as
    those that should be present.

8.  For licensed biological products, each lot of final product must be
    tested for identity, purity, and potency prior to clinical use.
    Please discuss what analytical tests and acceptance criteria could
    be applied to each of these parameters to provide reasonable
    assurance of adequate product performance *in vivo*.

    a.  Please identify the characteristics discussed under question 7
        that would be useful in developing such tests.

    b.  Given the capabilities and limitations of animal models
        discussed previously (Question 3), please discuss how these
        models may be used to provide data to support the *in vitro*
        characterization tests.

    c.  Please discuss biological activity assays that may be used to
        measure the potency of each product lot to ensure product
        consistency. Are methods based on determination of viable cells
        by dye exclusion (e.g., for cells used immediately after
        culture) or formation of colonies in soft agar (where time
        permits, e.g., if final product is cryopreserved) adequate? If
        not, please suggest appropriate alternatives.

9.  Many products in this category consist of cells within a biological
    or artificial matrix. What special considerations (e.g., mechanical
    testing, histological analysis, spatial distribution of gene
    expression, etc.) does this present for product characterization and
    specifications? Please discuss in terms of product safety, purity,
    identity, and potency.

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