# Presentation: 213758

## Preclinical Animal Models of  Cartilage Repair and Regeneration

- Matthew J. Allen, Vet. M.B., Ph.D.
- Associate Professor, Orthopedic Surgery
- SUNY Upstate Medical University,
- Syracuse, New York

## Overview	

- 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 efficacy
- Identification of the most appropriate animal model
- Each of the commonly used models has advantages and disadvantages
- 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 site

## 	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

- * Bellemans, 1999

## Surgical Access

## Mouse

| Advantages | Disadvantages |
| --- | --- |
| Controlled sources available | Tiny joints -- limited access and limited lesion size/number |
| Inexpensive to purchase and house | Cartilage is too thin to be useful as a surgical model |
| Immunocompromised models available for allogeneic and xenogeneic cell therapies   | Imaging is time-consuming and very expensive   |
| Transgenic and knock-outs available |  |

**Mouse**

## The Rabbit

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

**The Rabbit**

## The Dog	

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

**The Dog**

## The Sheep

| Advantages | Disadvantages |
| --- | --- |
| Relatively inexpensive to buy ($250) and house (<$10/week) | Variable quality, perhaps from 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 exercise on long-term studies | Early post-op rehab hard to control  |
|  | Fat pad can obscure joint |

**The Sheep**

## The Goat

| Advantages | Disadvantages |
| --- | --- |
| Relatively inexpensive to buy ($200) and house ($10-30/week) | Disease status (CAEV) can be 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 sheep |  |
| Arthroscopy feasible |  |

**The Goat**

## The Pig

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

**The Pig**

## The Horse

| Advantages | Disadvantages |
| --- | --- |
| Largest of the models – multiple large defects possible | Ethical concerns – companion animals |
| Aggressive loading conditions provide a stringent test | Variable sources, not usually purpose-bred for research |
| Cartilage thickness close to that of the human | Special facilities needed for 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 |  |

**The Horse**

## 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 times

## 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 critical
- 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 cartilage

## Scaling from Animals to Humans

**Scaling from Animals to Humans**

- Inter-species variations in many biological processes can be related to changes in body size
- What about cartilage?

- Kleiber, 1947

## Cartilage Thickness

- 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

**Scaling Defects**

- H    R      G    H      R     G

-   Human             Rabbit   	        Goat

-    Thickness        Density                                 Extent of the Defect

- 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 materials

## 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

**Surgical Technique -- ACI**

- www.carticel.com

## 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-differentiation
- 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 appealing
- 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 sites
- 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 therapies?
- 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 humans?
- 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*

## Summary

- 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