Preclinical Animal Models of Cartilage Repair and Regeneration

					Preclinical Animal Models of
    Cartilage Repair and

       Matthew J. Allen, Vet. M.B., Ph.D.
    Associate Professor, Orthopedic Surgery
       SUNY Upstate Medical University,
              Syracuse, New York
   Why use animal models to study cartilage repair?
   What is the question that is being asked
       Mechanistic vs. proof of concept vs. dose finding vs. safety and
   Identification of the most appropriate animal model
       Each of the commonly used models has advantages and
   Rational selection of outcome measures and time points
   Practical recommendations
       Initial screening
       Pivotal testing for safety and efficacy
   Limitations of current animal models
            Why Use Animal Models?

   Many key issues relating to chondrocyte differentiation
    and function can be studied in vitro
   In-vitro tests can also be used to characterize the
    interactions between cells and matrices
   Animal tests are however critical to explore
       cell growth and differentiation within an articular environment
       local and systemic safety
       stability of cell function, marker expression and mechanical
        properties of the neocartilage
       long-term effects of tissue remodeling within and around the repair
                      Purpose of the Study
   Mechanistic study of basic biological processes
   Preliminary dose-finding study
       physical intervention (e.g. drilling)
       cellular therapy
       gene or growth factor therapies
   “A versus B” study (comparison with predicate treatment)
       prove that it is at least as good as an existing therapy
   Pivotal safety and efficacy study for new treatment
       what constitutes efficacy?
       statistical versus biological significance
       accuracy, sensitivity and relevance of safety studies
               Available Models

 Rodent (mouse, rat)
 Rabbit
 Nonhuman primate
 Pig
 Small ruminants (sheep, goat)
 Horse
          Key Issues in Repair Models

   Age and its effect on skeletal turnover
   Bilateral versus unilateral surgery (IACUC issue)
   One- or two-stage surgery (IACUC issue)
   Nature of the implant
       Autogenous or allogeneic cells
       Composition of the scaffold
   Size (area, depth) and location of the lesion
       Partial or full thickness, critical or non-critical size
       Weight-bearing versus non-weight bearing location
   Appropriate surgical controls
       Same site, perhaps from the contralateral limb
            Which is the Best Model?
   “Best” should refer to relevance to humans but it is also
    impacted by practical considerations such as
       Cost (short studies in small animals are least expensive!)
       Availability of facilities, equipment and skilled personnel
   Comparative anatomy and joint function
   Surgical access to the articular surface
       Larger joints provide more cartilage
       Larger joints may also permit arthroscopic procedures
   Rehabilitation protocols
   Outcome measures
       In-life and post-mortem
   One model may not be ideal for every stage of testing
         Skeletal Anatomy
Human*      Goat    Sheep *                Pig *

Dog        Rabbit    Rat               Mouse

                       * Bellemans, 1999
Surgical Access

         Advantages                      Disadvantages
Controlled sources available     Tiny joints -- limited access and
                                 limited lesion size/number
Inexpensive to purchase and      Cartilage is too thin to be useful as
house                            a surgical model

Immunocompromised models         Imaging is time-consuming and
available for allogeneic and     very expensive
xenogeneic cell therapies
Transgenic and knock-outs
                          The Rabbit

         Advantages                          Disadvantages
Controlled sources available         Relatively small joints
Inexpensive to buy ($300) and        Thin cartilage
house ($20/week)
Easily managed within a              May heal “too quickly”
laboratory setting
Good surgical model with proven      Cannot use full-sized implants or
track record in bone and cartilage   fixation devices
                                     Loading conditions are not very
                                     aggressive - best suited for early
                                     testing (6-8 weeks)
                        The Dog

         Advantages                          Disadvantages
Controlled sources available          Ethical concerns -- companion
Arthroscopy feasible                  Expensive to buy ($800-$1250)
                                      and house ($50/wk)
Widely used in OA research            May be hard to obtain skeletally
(Pond-Nuki model)                     mature animals
Docile; tolerate casts and external
fixators so can do controlled rehab
                          The Sheep

         Advantages                         Disadvantages
Relatively inexpensive to buy       Variable quality, perhaps from
($250) and house (<$10/week)        commercial flocks
Range of sizes possible             Late skeletal maturity
Knee anatomy similar to humans      Disease status (Q fever, Toxo)
Arthroscopy feasible but not easy   Variable cartilage
Outdoor housing allows free         Early post-op rehab hard to control
exercise on long-term studies
                                    Fat pad can obscure joint
                             The Goat

          Advantages                        Disadvantages
Relatively inexpensive to buy       Disease status (CAEV) can be
($200) and house ($10-30/week)      problematic in some areas
Food and fiber animals              Can be tricky to anesthetize
Range of sizes possible             Late skeletal maturity
Knee anatomy similar to humans      Browsers by nature – can be hard
                                    to control post-op recovery
Cartilage relatively thicker than
Arthroscopy feasible
                               The Pig

          Advantages                      Disadvantages
Readily available (including      Rapid skeletal growth can be
minipigs)                         problematic (100 kg in 4 months!)
                                  so miniatures are preferred (<45
                                  kg by 4 months)
Better student and facility       Larger pigs can be noisy and
acceptance than domesticated      extremely hard to manage in all bit
animals                           a farm-type environment
Anatomy close to that of the
human – cardiac, GI tract, bone
Range of sizes possible
Can be arthroscoped
                          The Horse

          Advantages                        Disadvantages
Largest of the models – multiple    Ethical concerns – companion
large defects possible              animals
Aggressive loading conditions       Variable sources, not usually
provide a stringent test            purpose-bred for research
Cartilage thickness close to that of Special facilities needed for
the human                            surgery, imaging, necropsy –
                                     cannot use hospital scanner!
Arthroscopy and biopsy feasible     Personnel safety is an issue
Well characterized temporal
pattern of healing
Clinical need exists
                  Outcome Measures
   Clinical function
       force plate, pain scores, ROM etc.
   Visual assessment
       2nd look arthroscopy ± biopsy versus necropsy
   Imaging
       radiography and MRI (requires general anesthesia)
   Histology
       standardization of sampling and scoring schemes
   Mechanical testing
   Marker analysis
       biochemical analysis of synovial fluid
       molecular analysis of tissue samples
                   MRI and Cartilage

   Development of standardized MRI protocols for cartilage
    would be helpful in animal studies
       repeated measures in the same animal
   Newer techniques such as dGEMRIC appear to be very
    useful in humans but practical application in large animal
    models will be problematic
       High field strength magnet required
       Delayed analysis may necessitate unacceptably long anesthesia
                 Histological Analysis
   Two scoring schemes are widely used
       O’Driscoll
       Pineda
   Tissue sampling protocols should be developed
       These should focus on both the repair site and the “healthy” tissue
        around the repair
       Identification of the margins and the center of the original lesion is
   Contemporary (not historical) controls should be included
    whenever possible
   A combination of tinctorial and immunohistochemical
    staining provides maximum information
                 Mechanical Testing

   Compression testing
       Confined or unconfined

   Indentation testing
       Potentially useful in vivo as a means of assessing the repair
   As with all analyses, relevant controls are critical
   Ultimately, though, the properties of a “good” repair have
    not yet been fully defined
       assumption is that repair tissue should behave like hyaline
Scaling from Animals to Humans

                               Kleiber, 1947

 Inter-species variations in many biological processes can
  be related to changes in body size
 What about cartilage?
                             Cartilage Thickness
                                       Medial Femoral Condyle


Thickness, mm





                      Human      Horse       Goat      Sheep       Pig        Dog      Rabbit       Rat

                Based on the following sources:
                Changoor et al., 2004 (horse); Shepherd and Seedhom, 1997 (human); Jackson et al.,
                2001 (goat); Lane et al., 2004 (goat); Gelse et al., 2003 (rat); Oakley et al., 2004 (sheep);
                Hunziker and Rosenberg 1996 (rabbit, minipig)
Chondrocyte Morphometry

               Hunziker and Quinn, 2003
         Scaling Volumes and Areas

   Cartilage lesions represent a volume of tissue loss
   For a standardized lesion, healing potential should
    depend on the area (?)
   Given their inherent biological differences, animal
    models should only really be expected to provide relative
    (rather than absolute) information on healing rates for
    new therapies
                Scaling Defects

Thickness   Density               Extent of the Defect

H   R   G   H   R     G   Human       Rabbit             Goat

                            0%         95%               85%
                                  Marrow contact

                                   from Hunziker, 1999
      Location of Defects


Autologous Chondrocyte Implantation

   Modeling ACI generally requires two procedures
       initial tissue harvest
       implantation of cells with/without scaffold
   In inbred animals, an alternative would be to evaluate
    syngeneic cells
   One-step procedures are being evaluated in animal
    models (Bertone et al., 2005)
      cartilage broken up and seeded onto scaffold

           Autologous Chondrocytes

   ACI has been performed in
       Rabbits (Brittberg et al., 1996)
       Sheep (Russlies et al., 2005)
       Dogs (Breinan et al., 1997)
       Goats (Vasara et al., 2004)
       Horses (Litzke et al., 2004)
   Failures have been associated with
       Displacement of the periosteal patch
       Intrinsic problems with the animal models?
Surgical Technique -- ACI

          Autologous Chondrocytes

   Graft locations appears to have an impact, as in humans
   No clear guidance exists for lesion size, cell density etc.
   Studies should evaluate a range of cell doses since this
    is, in effect, what happens in clinical ACI
   Future work will focus on enhancing the proliferation,
    survival and/or function of transplanted cells
   In this context, ex-vivo gene therapy appears to offer
    tremendous potential
       Bone Marrow Stromal Cells

 BMSCs have the potential to differentiate into
  chondrocytes in vitro and in vivo
 Limited number of studies have been performed in
  animal models
    Rabbits (Diduch et al., 2000)

 Appealing concept since the cells can be expanded and
  manipulated ex vivo, prior to being re-implanted
 Potential concerns remain, including the potential for de-
 In animal models, BMSC collection may necessitate
  general anesthesia
                     Allogeneic Cells
   Genetically modified cells have been evaluated
    extensively in horses
       BMP-7, IGF-1, TGF-
   Application of allogeneic or xenogeneic cells is
       eliminates the need for two surgeries
       permits the use of cells from young donors
   Potential draw-backs are
       immunogenicity
       disease transmission
   Animal models must explore immune response as well
    as potential local/systemic toxicity of growth factors
                Osteochondral Grafts
   Studies in goats, sheep, dogs and horses all support the
    potential of autograft and allograft osteochondral transfer
    to repair lesions
   Concerns remain about
       Autograft donor site morbidity
       Long-term survival of implanted chondrocytes
           Snap frozen less good than cryopreserved
           In humans, viability decreases after 14 days of storage
            (Williams et al., 2003)
           Immune response to allograft OCT poorly understood
       Differences in the mechanical properties of cartilage from different
       Irregularities in the surface contour at the recipient site
OCT in the Goat

           *Lane et al., 1999
   The Limitation of Animal Models

“Although the repair of articular cartilage defects has been
studied in many species including rabbits, goats, and
sheep, there is no consensus on the most appropriate
animal model….”
“…none of these species replicate the anatomical, cellular,
and biomechanical properties of the human knee.
Therefore, we selected the most closely related species, a
nonhuman primate (NHP), that may exhibit a healing
response most similar to that of humans….”

                                          Gill et al., AJSM, 2005
          Current Recommendations

   Preliminary, short-term proof of concept can be
    performed in rabbits
   Definitive tests of efficacy in small lesions can be
    performed in the goat
       Feasible at most institutions
       Already established as the standard model

   Confirmatory studies of efficacy in large lesions can only
    really be performed in the horse
             Issues for Consideration

   What is the appropriate scaling factor for cell-based
   Are there inherent differences between chondrocytes
    from different species
       differences are known to exist between young and aged cells
       response to growth factors can be variable
   What is the ideal rehabilitation protocol? Is this practical?
       best for the animal vs. most relevant to humans
   Are clinical outcomes in animals predictive of those in
   How long do we need to follow animals?
                 Immune Responses

   Disruption of the subchondral bone permits vascular
    access and immune recognition
   Fresh osteochondral grafts are highly immunogenic
   Freezing reduces immunogenicity, probably by reducing
    the transfer or donor leucocytes
   Studies in dogs have documented both cell-based and
    humoral responses against MHC determinants
   Since immune responses are known to diminish long-
    term function, attention must be paid to evaluating these
    responses in vivo

   No single animal model is ideal for all stages of testing
    (this is often true)
   A rational approach would be to screen strategies in
    small animals and then confirm in large animals
   Emphasis should be on
       safety
       confirmation that the strategy is at least as effective as the
        current benchmark
   Ultimately, controlled clinical trials are needed to
    document long-term efficacy

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