ICRS 2019 - Conference Calendar
8.3.1 - Are Rodent Models Relevant?
Abstract
Introduction
According to dogma, cartilage has no or only limited regenerative potential, which would explain why initial cartilage damage (for example after joint injury) very frequently progresses to osteoarthriits. However, recent evidence suggest that cartilage indeed possesses at least some regenerative potential. Studies done in rodents, particularly in mice, suggest the presence of precursor cells within cartilage or surrounding tissues (e.g. the synovium) that can help regenerate damaged cartilage. This potential appears to be strain-specific, suggesting that it is regulated by genetic factors. Other factors such as age of animals are also influencing the efficiency of regeneration. This talk will review the current state of knowledge on cartilage regeneration, with particular focus on what we can learn from rodent models.
Content
Studies on cartilage regenration and in particular the underlying molecular mechanisms are almost impossible to do in humans since these studies a) need to be performed using in vivo models with the appropriate tissue interactions, biomechanical loads, oxygen tension etc., which are very hard to mimic in vitro; b) require the collection of biological samples from multiple time points in the process. Therefore, these studies are usually performed in animal models. In particular, mouse models are very useful for these studies due to our ability to manipulate the genome of mice. While CRISPR/Cas9 technology will ultimately allow similar approaches in other mammalian species, at this moment the tools available in the mouse are unmatched by other specieis. I will provide examples of how genetic engineering helps us to provide a better understanding of the molecular and cellular processes invovled in cartilage regeneration.
Using examples from several laboratories (e.g. Linda Sandell, Francesco Dell'Accio, and Roman Krawetz), I will first describe how the use of different mouse strains (all from the same species but with different genetic backgrounds) allows us to understand the large variability in regenerative potential, what the general contribution of genetic factors to this variability is, and how regeneration of articular cartilage correlates with regeneration of other tissues in the body.
I will then discuss an example (from Cosimo de Bari's lab) of how genetic labelling of cells allowed the identification of specific cell types within the joint that contribute to cartilage regeneration in an injury model. Identification of these cell types is crucial to develop strategies for manipulating them when we want to promote cartilage regeneration and endogenous repair.
Finally, I will provide a few examples from our own work, as well as from experiemtns performed by our collaborator Ling Qin, how genetic technology was used to remove or overexpress specific genes in the EGFR pathway in cartilage. These exeriments allowed us to identify a key role of this pathway in the growth (and potentially re-growth after injury) of articular cartilage.
These examples illustrate how we can use mouse models to better understand cartilage biology including regeneration. New insights from these studies will ultimately help to understand the endogenous repair of cartilage and to utilize these mechanisms for therapeutic purposes.
References
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Acknowledgments
I acknowledge the excellent work of my laboratory members and funding form the Canadian Institutes of Health Research and the Canada Research Chair Program.
8.3.2 - What is the Best Large Animal Model?
Abstract
Introduction
Large animal models for cartilage repairLarge animal orthopaedic models provide the opportunity to investigate the efficacy of novel therapeutics and the mechanisms of healing in animals that are more closely related in size, anatomy and physiology to man than rats and mice. However, there are many factors to consider before embarking on a large animal model, some of which will be considered in this talk. These include which species to use, which joint to use, which anatomical site within the joint to use and what size/shape/depth of defect to use? All of these different aspects of the animal model contribute to the validity of the model to be used and, ultimately, the reliability of the data generated.
Content
Which animal?In orthopaedic research, the choice of which animal to use is fundamental. The term ‘large animals’ refer to dogs, pigs, sheep, goats and horses, in contrast to ‘small animals’ which refers to rats, mice and guinea pigs. For the purposes of this talk, rabbit models are considered small animal models although some researchers consider them to be ‘large’, at least in relation to mice and rats. However rabbits have important physiological differences to ‘large animals’ and man, particularly in their enhanced healing abilities and will not be discussed further here.
The choice of animal species depends on many factors including the facilities and funding available to you, animal availability, ethical considerations and prior experience and expertise. For example, the use of dogs and horses in medical research is less socially acceptable in some cultures/countries than in others, despite their scientific merit, whereas sheep and goats are readily available and relatively cheap to use. Horses are considered by many to have the most appropriate joint physiology and anatomy to model human cartilage and osteochondral disease but are expensive and require specialist facilities and experience.
A final consideration in the choice of species to use is to consider the investigations that will form part of your experiment. One of the negative aspects of using large animals is that reagents, particularly antibodies for cell characterisation and immunohistochemistry, are not readily available. In this respect there are more reagents available for use in pig models than other species.
Which joint?
Which joint to use in the animal is a much easier question to answer than which species to use, as the majority of published orthopaedic studies are performed in the equivalent of the human knee (tibiofemoral and patellofemoral joints), which is correctly termed the ‘stifle’ of the animal. However, be aware that many studies erroneously refer to this joint as the ‘knee’ in animals, which, in quadrupeds describes the carpus! By conducting your experiment in a commonly used joint, experimental results can be more easily compared and contextualised. In addition to the use of the stifle, other specific anatomical models are described where the modelling is appropriate, for example rotator cuff models use the scapula-humeral joint and Achilles tendon repair models use the Achilles tendon.
Which anatomical site?
Having decided on the joint to use, the specific anatomical site within the joint needs to be considered. Specific parameters for consideration include whether the site is weight bearing, for example the femoral condyle, or not, for example the patella groove/trochlea sulcus. It has been known for many years that healing occurs at different rates and with different histological parameters in different anatomical sites within the same joint. Thus comparisons of defects at different anatomical sites within one experimental study may well not be valid and attention must be paid to the siting of the defect in the joint.
What size defect?
The final consideration when designing the large animal model is the size of the defect. A ‘critical sized’ defect refers to a defect that is too large to heal naturally. In a therapeutic trial, regardless of the nature of the therapy, using too small a defect will reduce the chances of observing and measuring a significant difference in healing. Conversely care must be taken not to create too large a defect that causes significant physical joint disturbance and the rapid onset of osteoarthritis – unless of course that is what you are modelling.
8.3.3 - α10hi MSCs Decrease Synovial Membrane Long Term Expression of TIMP-2 and NFκB Following Articular Injury
Abstract
Introduction
INTRODUCTION: Mesenchymal stem cell (MSC)-based therapies can limit the progression of focal cartilage lesions and prevent ongoing cartilage degeneration after joint injury by modulating the joint environment and/or contributing to repair. Integrin α10β1-selected mesenchymal stem cells (α10hi MSCs) are immunomodulatory, and display improved adhesion to osteochondral defects.1 In a previous study2, intra-articular administration of α10hi MSCs four days post-articular impact surgery exhibited protective effects against posttraumatic osteoarthritis (PTOA) in equine talocrural joints. To gain insight into mechanism, the current study aimed to determine if there was differential gene expression between treated and untreated joints. A NanoString custom codeset and nCounter SPRINT profiler was utilized to analyze mRNA extracted from formalin fixed paraffin embedded (FFPE) synovial membrane samples.
Content
METHODS: Adult horses (2-5 years, n=7) were anesthetized and three focal cartilage injuries were arthroscopically delivered on both tali. In each horse, joints were randomized to receive treatment with 20 x 106 α10hi MSCs (treated), or vehicle only (control). FFPE synovial biopsies from both tali were obtained from each horse at 3 timepoints: initial surgery, six weeks second look arthroscopy, and six months euthanasia post-injury. RNA was extracted from each sample using High Pure FFPET RNA Isolation Kit (Roche) and qualified for NanoString using NanoDrop, gel electrophoresis, and a BioAnalyzer smear analysis. To detect expression of 39 genes associated with early posttraumatic osteoarthritis (PTOA), a custom NanoString codeset was developed. For each sample, 200ng of extracted RNA was run against the codeset on an nCounter SPRINT profiler. NanoString nSolver 2.6 Analysis Software (NanoStringTechnologies) was used to process raw data. Background threshold was calculated from the raw data as the mean ±2SD mRNA count across all the negative controls. Data were normalized against three housekeeping genes (GAPDH, HPRT1, and UBC). Normalized log transform data were exported in JMP Pro 13 and analyzed using paired t-test, with p < 0.05 as significant.
RESULTS: We designed a custom NanoString gene expression panel, including a subset of 39 genes selected to describe innate and adaptive immune system signaling, GPCR downstream signaling, extracellular matrix organization and metabolism of protein. Six months after injury, synovium gene expression did not display a specific pattern associated with the treatment (Fig 1a), but CCL-5 expression was increased in the treated joints (p=0.028, negative binomial regression; Fig 1b). Interestingly, PRG4 expression had a trend toward higher expression in treated versus control joints (p=0.09). Paired t-test analysis (to account for the dependency of treated and control joints within the same horse) revealed that treatment with α10hi MSCs resulted in decreased expression of TIMP-2 (p=0.028) and NFκB (p=0.031) and increased expression of CCL-5 (p=0.049; Fig 2).
DISCUSSION: After traumatic injury, cartilage homeostasis is disrupted, and oxidative and inflammatory stresses deregulate gene expression involving metalloproteinase production leading to joint destruction. Abnormal NF-κB activation provokes production of pro-catabolic mediators inducing cartilage degradation3, while early suppression TIMPs seems predictive of therapeutic outcome4. The decreased expression of these two genes in joints treated with α10hi MSCs may be indicative of earlier recovery from the posttraumatic pro-inflammatory and catabolic state. MSCs are known to be a source of the chemokine CCL-5 in culture2 and the increased expression of CCL-5 in the joint treated with MSCs could indicate the persistence of such cells in treated joints. A trend toward higher PRG4 expression may explain previously reported increase in lubricin localized to areas of cartilage injury in α10hi MSC-treated joints.2
SIGNIFICANCE: Post-traumatic injection of α10hi MSCs could represent a therapeutic aid to modulate the signaling pathways activated from the trauma which lead to chronic cartilage degradation.
References
REFERENCES: 1) Lundgren-Akerlund+ ICRS (2018). 2) Delco+ OAC (2018). 3) Karnoub+ Nature (2007). 4) Olivotto+ RMD Open (2015). 5) Rooney+ ARD (2010).
Acknowledgments
Funded by Xintela AB, The Harry M. Zweig Fund for Equine Research, and NIH/NIAMS Mentored Clinical Scientist Career Development Award 1K08AR068470 (MLD)