Short summary

Osteoarthritis (OA) is the main reason for chronic pain and disability worldwide. This causes a significant impact to the public health system in every developed country, where OA currently affects one in eight individual. The main cause for OA development is joint-affecting trauma. Despite adequate restoration of anatomy, the risk to develop a posttraumatic osteoarthritis (PTOA) after 10 years is higher than 40. Intraarticular catabolic conditions following fractures are mainly triggered by pro-inflammatory cytokines. These are supposed to be responsible for the degradation of cartilage matrix.

Aim of the project is the in-vivo identification and in-vitro inhibition of cytokine cascades leading to the initial events of PTOA as breakdown of extracellular matrix and chondrocyte apoptosis. 

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Rationale

Osteoarthritis (OA) affecting one in eight individuals is the main reason for chronic pain and disability worldwide. The main cause for OA development is joint-affecting trauma and risk factors have primarily been associated to previous biomechanical treatment. Therefore, the principle of current fracture treatment is anatomical reconstruction of the joint surface combined with functional and adequate aftercare. However, despite correct restoration of anatomy and application of sufficient physiotherapy, the risk of posttraumatic OA remains as high as 40%.

Joint trauma initiates an inflammatory cascade leading to synovial catabolism and cartilage degradation, a fact, which to date has been ignored in standard therapy. Unfortunately, due to the lack of blood supply, cartilage regenerates much less efficient compared to bone.

Previous studies suggest that the synovial biochemical milieu may be of decisive importance for chondrocyte and cartilage survival or degeneration. We therefore hypothesize, that protecting cartilage and chondrocyte by inhibiting the post-injury inflammatory cascade, might contribute to durable successful results in fracture therapy. Consequently, the aim of the current project is to identify the cytokines initiating the inflammatory cascade. Moreover, we aim to clarify how to inhibit the cytokine cascade and thus prevent the development of posttraumatic OA. In specific, we intend to answer the following research questions:

• Are there differentially regulated synovial pro-inflammatory proteins in ankles with and without a fracture?
• Do the identified and relevant markers/cytokines correlate with short and middle term clinical symptoms?
• Does the inhibition of pro-inflammatory cytokines prevent cartilage destruction and chondrocyte death in-vitro?

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Description of the cohort

The scientific approach will include two parts, the in-vivo analysis of biomarkers in combination with clinical data and the test of inhibitory strategies by means of an in-vitro model. Ankle fractures are an attractive clinical model for this research question, because they have a high caseload, reliable control groups, validated procedures for sample taking and preliminary data for comparison. The clinical part is carried out in the Department of Orthopedic Surgery at Odense University Hospital, Svendborg, while biomarker analyses mainly take places in the Molecular Endocrinology unit (KMEB), Department of Endocrinology, Odense University Hospital. 

Identification of differentially regulated synovial pro-inflammatory proteins in ankles with and without a fracture.

Effusions are collected during a clinical trial comparing not injured joints with intraarticular ankle fractures. Technically, specimens are obtained by anterolateral puncture of the ankle joint prior to the operation as described and validated in our previous work. Control samples are obtained from osteochondritis dissecans (OCD) type 2 patients, undergoing arthroscopy. In these patients, the cartilage is intact, indicating a nearly healthy joint without inflammation. Secondly, effusions from patients undergoing an implant removal are collected. Samples from control patients are collected without additional risk to the patients. As described, the comparison of the different cytokine levels is based on corrections using the total protein content.

To reach a power of 80 %, we include 62 patients in our study, and based on previous studies we include 10 patients in each control group.

Inclusion criteria: Existence of an acute fracture involving the ankle (location AO 44) requiring open or closed reduction and internal or external fixation within 14 days; Age between 18 and 65 years; 

Being able to read and understand Danish or English; Informed consent.

Exclusion criteria: Open fractures; Associated arterial and nerve injuries; Multiple injured patients with an Injury Severity Score more than 16; Primary or secondary infections; Injuries associated to a Charcot foot.

The following epidemiological parameters are recorded: age, sex, BMI, classification of injury according to AO standards, allowed weight bearing. Aftercare follows the Odense guidelines with a static walker.

Patients are evaluated 3, 6, and 12 months post injury according to the following clinical parameters: Pain (visual analog scale), return to work (days), swelling (measurement of circumference at malleoli (cm)), effusion and synovitis (clinical evaluation), and validated scores (the American Orthopedic Foot and Ankle Score, the Foot Function Index - DK, the Euroqol 5D questionnaire ). 

For the discrimination between the fracture and the control groups, a proteomics analysis is used. This allows the identification of innovative biomarkers and starting points for in-terventions. To assess the proteomic differences, specimens from the control groups and the fractured joints are subjected to 2D-LC?MS/MS and 4-plex iTRAQ-based quantitative analysis. 

Besides proteomics, three classical levels of analysis of effusions are applied to facilitate the discrimination of the acute fracture group and the controls. These are: measurement of protein-markers in effusions, analysis of the cellular composition, and synovial histology. 

Using specific ELISAs, classical pro-inflammatory cytokines (as IL -1alfa/beta, IL-6, IL-8, acti-vated complement components), extracellular breakdown products (aggrecan, collagen type 2 cleavage products) and typical cartilage metabolites (as bFGF, BMP-2, BMP-7, IGF-1) are determined and mark the inflammation and cartilage turnover. 

Collaborating researchers and departments

University of Southern Denmark

• Consultant and professor Moustapha Kassem

Department of Orthopedics, Odense University Hospital

• Consultant Johnny Frøkjær
• Consultant Lonnie Froberg, PhD
• Associate professor Kate Lambertsen
• Associate professor Lars-Henrik Frich 
• Consultant and professor Jens Martin Lauritsen

Department of Orthopedic and Trauma Surgery, University of Freiburg, Germany

• Professor Anke Bernstein