Detection of Bacterial DNA and Collagen Metabolism in Acutely Ruptured Achilles Tendons

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 The findings were based on the 14C bomb pulse method, and unpublished data from the same re-search group (using this method) indicates that an abnormally high rate of collagen turnover precedes symptoms of tendon overuse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