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Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
Canadian Orthopaedic Foundation Grant Application
Title: The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
Grant Amount Requested: $10,000
Research start date: 2007/07/01
Research end date
: 2008/06/30
Location of Conducted Research: Joint Injury and Arthritis Research Group (JIRG),
Health Sciences Center, Faculty of Medicine, University of Calgary.
Applicant
: Michael Monument, MD – Orthopaedic Surgery resident; resident member of
the COA
Supervisor: Kevin Hildebrand, MD, FRCSC – Upper Extremity Surgeon; member of the
COA
Collaborator: David Hart, PhD
Age of Applicant: 26 (Michael Monument; 1980/09/15)
Objective/Methodology/Relevance to Orthopaedics: please see attached document
Subjects: Rabbits; no humans. Study proposal has been approved by the University of
Calgary animal ethics committee.
Is the research ongoing? No. Experiments will commence July 2007.
Letter from section-head: Dr. Cy Frank, Head of UofC Orthopaedics confirming
originality of proposed research is attached.
We have not previously applied to the COF for funding of this project.
Previous funding by the COF:
Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
Other funding sources:
• University of Calgary Graduate Studies internal funding – status: pending.
• University of Calgary, Division of Orthopaedics internal funding – status: Funds Administration: Funds will be administered through a University of Calgary trust
account, under Dr. Kevin Hildebrand.
• Contact Info:Dr. Kevin HildebrandDepartment of Surgery3330 Hospital Drive NWCalgary, ABT2N 4N1 Office phone: (403) 220-7282Office fax: (403) 270-3679hildebrk@ucalgary.ca Project Budget: please see attached word document.
Non-technical summary: please see attached word document
In closing, thank you for taking the time to review and consider this application and we hope this project strikes you with same interest and enthusiasm as it does within our laboratory.
Orthopaedic SurgeryFaculty of MedicineUniversity of Calgary Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
Technical Project Summary
Title: The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
Introduction and Orthopaedic Relevance: Post-traumatic joint contractures represent a
common and difficult problem facing musculoskeletal health practitioners. Post-
traumatic joint contractures can occur in numerous joints such the shoulder, knee, ankle
and the hand. The elbow is of particular interest as it is extremely vulnerable to post-
traumatic joint contractures, even after very brief periods of immobilization.
Contractures of the elbow can be painful and the resulting stiffness and motion loss can
impede ones activities of daily living such as eating, drinking and clothing. A flexion-
extension arc of 30° - 130° is needed for activities of daily living and roughly 5% of
elbow injuries will not regain functional ROM, even after aggressive conservative
therapy. In a recent review of the Calgary Health Region from 2002 – 2005 revealed
1200 elbow dislocations or fractures during that period, further emphasizing the clinical
impact of post-traumatic joint contractures in our society.
Numerous intra-articular pathologies have been attributed to the development of post-traumatic joint contractures of the elbow such as muscle-tendon shortening, joint incongruity, osteophytes and progression of post-traumatic arthritis. Yet in the setting of an anatomically reduced elbow, joint capsule fibrosis and hypertrophy has been identified as a significant and reliable causal agent in the genesis of stable contractures. Current surgical practice is to treat these elbow contractures by excising or dividing the fibrotic joint capsule. Surgical treatment does result in restoration of motion, but not all patients regain their functional range of motion, while others go on to relapse after a period of resolve, not to mention the acquired surgical risk with any invasive procedure to the elbow. In light of this, a treatment directed at preventing the formation of joint capsule fibrosis after a traumatic injury to the elbow would provide a useful adjuvant therapy to this troubling condition.
From a cellular level, joint capsule fibrosis has been paralleled to numerous other fibrotic diseases of the human body such as pulmonary firbrosis, liver fibrosis, adhesive capsulitis of the shoulder, hypertrophic wound healing and Dupuytren’s contracture of the hand. Common to all of these fibrotic processes is the overabundance and activation of myofibroblasts. The myofibroblast, a normal cell of connective tissue, is a differentiated lineage of the fibroblast, activated during the normal process of connective tissue healing. At the cellular level, the myofibroblast is exerts a mechanical contractile force on it’s surrounding tissues, while secreting various collagens, fibronectins and proteoglycans to reorganize the contracted matrix into a stable configuration. In the aforementioned pathologic states of fibrosis, the connective tissues are populated with a stable overabundance of myofibroblasts. Capsules harvested from the elbows joints of human subjects with significant post-traumatic contractures demonstrate an identical pattern of Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
myofibroblast over abundance compared to controls and the collagen composition of the capsules is also markedly different.
The implication of the myofibroblast in the genesis of fibrosis has stimulated great interest in the determination of factors involved in their differentiation and activation. Mast cells have received great attention in this regard as many of the mediators synthesized and liberated by mast cells can positively influence myofibroblast development. In vitro studies have demonstrated the following: increased myofibroblast differentiation when fibroblasts are co-cultured with activated mast cells, histamine and tryptase can stimulate fibroblast differentiation, myofibroblasts and mast cells exists in close cellular contact and finally, histam ine stimulates collagen synthesis from myofibroblasts. Furthermore, mast cells are a normal resident synovial tissue and the joint capsule and an integral component of an inflammatory response, as in the case of musculoskeletal trauma. The synthesis of above information has lead our laboratory to hypothesize that the mast cell may be a crucial link in the sequence of events leading to a stiff, fibrotic, post-traumatic joint contracture. To test this hypothesis, we have developed an animal model of a post-traumatic joint contracture in the rabbit knee. Using this model, previous studies have demonstrated many similarities at the cellular and extra-cellular level to that noted in other models of fibrosis: in particular elevated myofibroblasts numbers, elevated levels of the contractile protein - α sma and altered collagen compositions and synthesis compared to control. These findings have paralleled the changes noted in capsules taken from the post-traumatic contractures of the human elbow. By using a systemic mast cells stabilizer and histamine receptor antagonist Ketotifen fumurate, our next series of experiments will attempt to target the role of the mast cell in the genesis on a post-traumatic knee joint contracture in our rabbit model.
Hypothesis 1: Mast cell numbers will be increased in capsules harvested from post-
traumatic joint contractures when compared to capsules from joints free of contracture.
Hypothesis 2: Inhibition of mast cell degranulation using a systemic mast cell stabilizer,
Ketotifen Fumarate, will lessen the degree of post-traumatic joint contracture in our
animal model.
Hypothesis 3: Mast cell inhibition using Ketotifen fumurate will decrease the absolute
and relative count of myofibroblasts and the production of -
Hypothesis 4: Mast cell inhibition will also decrease the metabolism and turn over of the
contracted joint capsule matrix represented by decreases in the synthesis of collagen
types I, II, III, V and MMPs.
EXPERIMENTAL DESIGN:
Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
One large, multi-dimensional series of experiments will be used to test the above hypotheses. As mentioned above, a rabbit model representing a stable post-traumatic knee joint contracture will be used in these experiments. In brief, stable post-traumatic knee joint contracture will be created surgically via the creation of a intra-capsular, yet extra-articular fracture in the distal rabbit femur, followed by surgical fixation and immobilization of the knee in 150° of flexion for eight weeks. Eight weeks of immobilization has been previously demonstrated to create a sustained and stable degree of contracture in the rabbit knee, even after re-mobilization up to 32 weeks.
After eight weeks of immobilization, all rabbits will be sacrificed and surgical knees as well as contralateral control knees will be bio-mechanically analyzed for degrees of joint contracture. An identical numbered group of non-surgical rabbits will also be used as a control for all measures and subsequent testing.
Following the biomechanical measurements, posterior joint capsules of both surgical and contralateral knees and as well non-surgical control rabbit knees will be harvested for appropriate immunohistochemistry and cellular investigations. Mast cell and myofibroblast counts will be made using immunohistochemistry staining with anti-tryptase and anti-α-SMA of harvested capsule sections.
Western blots will be performed to quantify - α SMA, chymase, tryptase, various collagen types, MMP and TIMP protein levels extracted from our experimental and control posterior joint capsule tissue.
A total of three study arms will be needed: the first (control arm) will consist of rabbits untouched by any surgical or pharmacological intervention. The second arm (experimental #1) will have the right knee surgically manipulated and subsequently immobilized for a total of eight weeks. No pharmacologic interventions will be given to this study arm. The third study arm (experimental #2) will have the identical surgical intervention as experimental #1, but in addition to this, this group will receive a twice daily dose of Ketotifen Fumarate (KF), a potent mast cell stabilizer throughout the entire eight weeks of immobilization. All rabbits will be sacrificed exactly eight weeks from the initial day of surgery and all surgeries will take place over the duration of two days. The non-surgical group will be arbitrarily assigned a start date given their non-surgical treatment.
Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
• Rabbits: $110/rabbit + GST (30 rabbits; 20 experimental and 10 control) • ARC (Animal Resource Center) charge: $2.00/day (9weeks [8weeks for experiment & 1week for pre-conditioning] = 63days X 30 rabbits) $2.00 x 30rabbits x 63days = $3,780 ARC surgery: $250/set-up; 1set-up/3surgeries 20 surgeries x $250/set-up x 1set-up/3surgeries = $1,750 Euthanyl, Duplocillin, Torbugesic, Suture, Scalpels and K-wires.
TO TAL RABBIT CARE AND SURGERY = $8,193.25
KETOTIFEN FUMURATE (NovoPharm): $1.00/1mg 10mg/day x 10rabbits x 56days x 1.06 = $936.00 MOLECULAR TESTING (represented per joint capsule; there will be a total of 30 joint capsules studied in this section) • Western Blots (This includes primary and secondary antibodies for collagen I, II, III, and V; MMP-1, -2, -9, -13 and -15; and α-SMA). Estimated cost: $189.25/capsule x 30capsules = $2,677.39 • Immunohistochemistry (The cost of the primary antibodies to - chymase, and appropriate secondary antibodies). Estimates from previous experiments in our laboratory: $29.87/capsule x 30capsules = $896.00 TO TAL MOLECULAR TESTING: $3,573.39
BIOMECHANICAL TESTING: Biomechanical testing costs $60/hr and it takes about 1 hour to test one rabbit. 30rabbits x$60/hr = $1,800 TO TAL PROJECT BUDGET: $15,753.70
Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
Non-technical Project Summary
Significance
: Joint contractures represent a common and troublesome musculoskeletal
pathology encountered by health practitioners. Of particular interest, the elbow joint is frequently injured and quite vulnerable to the formation of disabling and painful post-traumatic contractures. Roughly 5% of elbow injuries will never regain functional range of motion. Joint capsule fibrosis has been identified as a major causal agent of post-traumatic joint contractures, yet a limited understanding of the capsular biology involved in the genesis of post-traumatic joint contractures exists. Background
: A great deal of similarities at the cellular level have been observed
between the fibrotic capsules of contracted joints and other fibrotic diseases involving various organ systems such as pulmonary and liver fibrosis or hypertrophic wound healing. The common link between these conditions is the over abundance of myofibroblasts within these tissues. Myofibroblasts are a normal cellular resident of connective tissue and represent a differentiated lineage of a ubiquitous connective tissue cell line, the fibroblast. In post-traumatic joint contractures myofibroblasts accumulate in pathologic proportions and mechanically contract their surrounding tissues. This has been demonstrated in contractures of human elbows and shoulders and the knee joint capsules from a proven rabbit model of post-traumatic joint contractures. What we don’t know is what mechanisms exist to stimulate this pathologic differentiation and accumulation of myofibroblasts with in these joint capsules. One possible link is the mast cell, a unique cell comprised of pre-formed inflammatory granules capable of liberating multiple compounds into the local cellular environment when stimulated. Studies suggest that the compounds liberated from these granules can stimulate myofibroblast differentiation. Mast cells are also normal residents of joint capsules and can easily be triggered by the inflammatory cascade accompanied with bony trauma. Hypothesis: The systemic inhibition of mast cell degranulation after a traumatic bony
injury to a joint will impede the genesis of joint capsule fibrosis by preventing mast cell
induced myofibroblast differentiation, resulting in clinically improved joint mobility.
Study Design
: A series of experiments will be carried out in a previously described,
rabbit model of a stable post-traumatic knee joint contracture. To reproduce joint injury, intra-articular fractures will be surgically created in skeletally mature rabbits followed by eight weeks of immobilization. Three study arms will be used: animals without injury or immobilization, animals with joint injury and immobilization and finally animals with joint injury, immobilization and systemic treatment with a mast cell stabilizer, Ketotifen fumurate during the immobilization period. At eight weeks, all animals will have the knee joint motion biomechanically measured and joint capsules will be harvested to quantify cellular, protein and RNA compositions of the three different experimental capsules. A total of thirty rabbits will be needed for ten subjects in each treatment arm.
Timeline
: Experiments will begin in August 2007, joint capsule harvest and joint motion
measurements will take place in October and capsule molecular tests will be carried out Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
in November and December. Data analysis and preparation will follow in January and February and thesis preparation and presentation leading into April and May 2008.
Michael MonumentSupervisor: Dr. K. Hildebrand The Role of the Mast Cell in the Creation of Post-traumatic Contractures of the
Rabbit Knee
Letter of endorsement from department head: Dr. Cy Frank is attached
with email in PDF format.

Source: http://www.canorth.org/Assets/pdfs/Monument%20Research.pdf