Detection of Bacterial DNA and Collagen Metabolism in Acutely Ruptured Achilles Tendons
Condition: Achilles Tendon Rupture
MeSH terms: Rupture
Study type: Observational

Summary: 
      The etiology and pathogenesis of acute Achilles tendon ruptures are complex and not fully
      understood. It is well known that they are associated with pre-existing pathological
      alterations, similar to the changes observed in tendinopathy.

      The present study investigates if bacteria and collagen metabolism play a role in the
      etiology of acute Achilles tendon rupture. During surgery, 20 patients will have taken two
      biopsies from the ruptured part of the tendon and two biopsies from the healthy tissue of the
      same tendon 2-4 cm proximal to the rupture, as a control.
    

Detailed description: 
      Objectives

        1. To investigate the collagen turnover in Achilles tendons prior to a rupture.

        2. To investigate the relative collagen turnover in Achilles tendons the days immediately
           after a rupture.

        3. To investigate the acute collagen protein synthesis rate in acutely ruptured Achilles
           tendons.

        4. To investigate if bacterial DNA is present in samples from the degenerative tissue in
           acutely ruptured Achilles tendons compared with samples from the healthy tissue of the
           ruptured tendon.

      Background

      Tendon ruptures are severe injuries that potentially lead to reduced function, reduced
      activity and sick leave. In younger, sports active individuals with strong tendons, an acute
      traumatic event is usually needed to trigger a rupture. With age the tendons are exposed to
      degenerative changes and can therefore rupture in everyday activities (spontaneously).

      The etiology and pathogenesis of spontaneous tendon ruptures are complex and not fully
      understood. It is well known that they are associated with pre-existing pathological
      alterations, similar to the changes ob-served in tendinopathy. Recently, Dakin et al. found
      that ruptured Achilles tendons show evidence of chronic (non-resolving) inflammation. The
      condition might be a result of collagenolytic injury followed by failed tendon healing. Yet,
      other theories are still highly debated.

      Many predisposing factors have been proposed to contribute to the pathological alterations of
      tendons. Studies found that factors such as increasing age, dominant arm, history of trauma
      and specific acromial anatomy are associated with rotator cuff tear. It is suggested that
      spontaneous tendon ruptures, primary Achilles tendon, are related to systemic and injectable
      steroids, fluoroquinolone use and some rheumatic diseases. Furthermore, studies have shown
      possible associations between genetic factors and tendon injury. The mentioned factors may
      influence the initiation and development of the patho-logical alterations that weaken the
      tendons. However, it is certain there are other mechanisms and predis-posing factors that
      need to be investigated.

      Heinemeier et al. found that the bulk of the collagen matrix of healthy human Achilles tendon
      core is an essentially permanent structure that is laid down during height growth and has
      limited turnover in adults. The findings were based on the 14C bomb pulse method, and
      unpublished data from the same re-search group (using this method) indicates that an
      abnormally high rate of collagen turnover precedes symptoms of tendon overuse
      (tendinopathy)It is of great interest to investigate if increased collagen turno-ver
      predisposes to tendon rupture.

      Recently, Rolf et al. demonstrated the presence of bacterial DNA in 25% of samples from
      ruptured Achilles tendons. Polymerase chain reaction (PCR) was used to identify the highly
      conserved bacterial 16S rDNA gene in the tendons. This finding open the question, if
      bacterial depositions play a role in the etiology of spontaneous tendon ruptures In order to
      prevent tendon ruptures it is essential to improve our understanding of the etiology. A
      better understanding may also lead to development of new therapeutic options.

      In this cross-sectional study, we aim to investigate if bacteria potentially are contributing
      to the etiology of acute Achilles tendon rupture, Additionally, we also investigate if tendon
      ruptures are preceded by an in-creased collagen turnover.

      Design of the study

      The study is conducted as a cross-sectional study. Patients with acute rupture of the
      Achilles tendon are included The study aims to include 20 patients with acute Achilles tendon
      rupture.

      The procedure for the patient:

        1. At the day of inclusion, the patients will have taken a blood sample and ingest 150ml of
           deuterium oxide (D2O).

        2. At the day of the surgery, the patient will meet 3 hours before the beginning of the
           surgery. A bolus of stable isotope (15N marked proline tracer) will be injected and
           afterwards continuous infused trough the antecubital vein. Blood samples are taken
           before infusion starts, 30min after, 60min af-ter, 120min after and when the biopsies
           are taken.

        3. During the surgery, the patients will have taken:

             1. Two biopsies from the stump end of the ruptured tendon or ligament.

             2. Two control biopsies 2-4 cm proximal to the stump end of the ruptured Achilles
                tendon.

      Procedure for biopsies

      All biopsies are taken by trained orthopedic surgeons. Every biopsy is taken during surgery
      in an operating theatre. Before the beginning of the surgery a sterile table is prepared
      containing three packs of sterile scalpels and forceps, and 8 containers for biopsies with
      the patient's social security (CPR), the type of analysis and a continues number individually
      describing each biopsy.

      The 8 containers will hold:

        1. Biopsy from the stump end of the ruptured tendon for 16S rDNA analysis.

        2. Biopsy from the stump end of the ruptured tendon for 14C bomb pulse method

        3. Biopsy from the stump end of the ruptured tendon for isotope analysis (deuterium oxide
           and 15N marked proline).

        4. Biopsy from the stump end of the ruptured tendon for mRNA analysis.

        5. Biopsy from the ruptured tendon 2-4 cm proximal to the stump end for 16S rDNA analysis.

        6. Biopsy from the ruptured tendon 2-4 cm proximal to the stump end for 14C bomb pulse.

        7. Biopsy from the ruptured tendon 2-4 cm proximal to the stump end for isotope analysis
           (deuterium oxide and 15N marked proline).

        8. Biopsy from the ruptured tendon 2-4 cm proximal to the stump end for mRNA analysis

      Biopsies from ruptured tendons

      Achilles tendon:

      The biopsies are taken during open surgery. To prevent contamination of the biopsies the
      following proce-dure is followed:

        1. Surgical rub and draping according to normal guidelines.

        2. Skin incision and dissection according to normal guidelines.

        3. As the ruptured tendon is identified, a new pack containing a sterile scalpel and a
           forceps is opened. The instruments are only used for taking the biopsy and does not get
           in contact with the surrounding tissue.

        4. 2 biopsies, 10mm long, 1mm broad and 1mm deep, are taken from the degenerated tendon
           tissue at the rupture site.

        5. The first biopsy is placed in a container (for bacterial detection). On a sterile board,
           the second bi-opsy is divided in three and placed in three different containers (one for
           14C bomb pulse, one for isotope analysis (both deuterium oxide and 15N marked proline)
           and one for mRNA). These contain-ers are frozen down to -80oC.

        6. A second new pack containing a sterile scalpel and a forceps is opened and 2 biopsies 10
           mm long, 1mm broad and 1 mm deep are taken 2-4 cm proximal to the rupture site.

        7. The first biopsy is placed in a container (for bacterial detection) On a sterile board,
           the second biopsy is divided in three and placed in three different containers (one for
           14C bomb pulse, one for isotope analysis (both deuterium oxide and 15N marked proline)
           and one for mRNA). These contain-ers are frozen down to -80oC.

      Contamination tests

      PCR and sequencing - Both a negative and a positive control is included in the test. Numbers
      of cycles in positive samples will also be included in the interpretation. Exclusively
      environmental bacterial species never been related to human disease are likely to be regarded
      as contaminants.

      Blood culture - During surgery, blood a sample is send for culture to investigate if bacteria
      were presented in the blood.

      Bacterial cultures - Bacterial culture tests are conducted on all patients. The investigated
      samples are taken with swaps from:

        -  Patients skin area over the surgical site.

        -  The blade of the used scalpel.

      The swaps are collected in separate glass tubes and send to Department of Microbiology,
      Hvidovre Hospi-tal. The swaps are tested on multiple culture media for multiple species.

      Blinding of the analysis

      For each included patient, the 2 containers with biopsies for bacterial detection will get a
      randomized continues number. These numbers can, via a key document (paper form), reveal if
      the biopsy has been taken from the degenerative tissue or from the healthy tissue. The person
      responsible for the analyze of the bacterial DNA has no access to the key document.

      Data registration during inclusion

        -  Patient ID

        -  Social security number

        -  Phone number

        -  Age

        -  Weight

        -  Date of examination

        -  Date and time of insult

        -  Corticosteroid injection (number and time)

        -  History of infection
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        Patients diagnosed with acute Achilles tendon rupture, seen in the outpatient clinic at
        Copenhagen University Hospital, Hvidovre.
      
Biospecimen description: 
      Biopsies from acutely ruptured Achilles tendonds will be taken, Two biopsies from the
      ruptured part of the tendon and two biopsies from the healthy tissue of the same tendon
      proximal to the rupture, as a control.