Arginine and nitric oxide (NO) metabolism during bone healing and non-union development - early prognostic markers

The incidence of hospital admission in the Netherlands due to a fracture is
50,000 per year, of which about 28,000 are operated upon. This incidence is
rising in the last decades due to increased aging of the population and
increased mobility of this group. Increased duration of hospital stay has a
major impact on medical and hospital resources and adequate recovery of the
patient with a normal fracture healing is, therefore, essential.
A fracture of the bone implies loss of mechanical integrity and continuity of
the bone.
This will induce a process of bone healing, which will restore the integrity
and continuity, when a number of conditions are met: absence of movements in
the fractured bone parts, absence of distraction of the fracture, and adequate
blood supply to the fracture site. Internal factors during this healing process
are bone growth factors and absence of osteoporosis and external factors are
adequate tissue coverage of the bone and adequate nutritional status.
Failure of bone healing, or non-union, occurs as a consequence of a standstill
in the healing process. A non-union can be defined as a fracture which has
ceased to show any evidence of healing, as indicated by persistent fracture
lines, sclerosis at the fracture ends, a gap, and hypertrophic or absent
callus. A non-union, which develops despite callus formation, is called
hypertrophic non-union, in contrast to atrophic non-union during which no
callus formation is seen. In general terms, unless bone loss is present, a
non-union is usually present when consolidation of the fracture has not
occurred 6-8 months after the occurrence of the fracture.

Arginine, a semi-essential amino acid, plays an important role in the
transport, storage and excretion of nitrogen. Moreover, arginine is an
essential factor in the metabolism, since it is a precursor of nitric oxide
(NO), polyamines and other molecules.NO is formed from arginine by the
expression of the enzyme NO synthase (NOS).Some studies indicate that NO has a
biphasic or dual effect on bone healing. High concentrations of NO inhibit bone
resorption by suppressing osteoclast formation and the resorbing function of
osteoclasts. Low concentrations of NO augment cytokine-induced bone resorption.
In line with these data an inhibition of growth and differentiation of
osteoblasts by high concentrations of NO was found, where low concentrations
regulate normal growth and function of osteoblasts.
Preliminary data from our PILOT experiment (MEC04-021) concerning the influence
of arginine, NO or polyamine metabolism on the development of non-union,
indicate that a non-union possibly occurs during a disturbed production of NO.
In the PILOT phase, bone callus and plasma amino acid concentrations were
evaluated to investigate disturbances in the amino acid profile during the
development of a non-union. Patients with either atrophic or hypertrophic
non-union had completely different amino acid concentrations of arginine,
citrulline and ornithine. The patients with an atrophic non-union showed an
overall decrease in the bone callus concentrations of arginine, citrulline and
ornithine, compared to control samples, as the hypertrophic non-unions showed
the opposite phenomenon with increased concentrations of these amino acids in
callus tissue. In our PILOT study (MEC 04-021), the disturbances in the
arginine-NO metabolism were determined at one time point, after the development
of a non-union, and not immediately after the onset of the fracture. During
fracture healing, fluctuations in the arginine-NO metabolism are possible,
combined with the changes in the NOS expressions during healing. Therefore,
early and frequent determination of the arginine -No metabolism in the
different phases of fracture healing are essential to gather insight in the
role of arginine-No in the development of a non-union. Furthermore, the
different measurement time points of the arginine-NO metabolism, may be used as
diagnostic time points, for the start of future therapeutic interventions to
prevent the development of a non-union. Prior to this, early detection, during
the healing process, of abnormal levels of arginine is a prerequisite for this
intervention.
Bone turnover rates play a role in the healing process of fractures. A
disturbed bone turnover may be a factor influencing the development of
non-unions. Developing a method with which the turnover rates of bone can be
measured adequately is therefore of importance.

Primary Objective:
To study the arginine-NO metabolism during fracture healing and dysfunctional
fracture healing

Secondary Objective(s):
To investigate if differences or decreased arginine and NO concentrations in
bone healing form a prognostic marker for non-union development

Research questions
1. What are the changesof the arginine and NO metabolism in the onset of
fracture healing in humans?
2. Can disturbances in the arginine and NO concentrations be used as prognostic
markers for non-union development or for normal fracture healing?
3. Are there fluctuations present in the arginine - NO metabolism during
fracture healing, which may be used as therapeutic intervention points in
future research?


Hypothesis
Early detection of disturbances in the Arginine and nitric oxide metabolism
during fracture healing are a good prognostic marker for non-union development

Prospective observational study.
Duration: 2-year period


This study is conducted in all acute fracture patients, with a fracture of the
tibia or femur attending the Department of General Surgery, to investigate the
Arginine -NO metabolism during normal fracture healing and possible
dysfunctional healing.
It is anticipated that changes in the arginine-NO metabolism occur rapidly
after the fracture onset, indicating that patients need to be included as soon
as possible.

The experimental design of the study is described below
1. After inclusion, baseline demographic details are registered, including
Injury Severity Score.
2. During the surgical procedure the bone pulp during intramedullary reaming
will be collected and snap frozen in liquid nitrogen. This reaming and thereby
harvesting of bone pulp is part of the normal procedure during fracture
osteosynthesis.
3. One blood samples (5 mL venous blood) will be collected to measure amino
acid levels in plasma of the patient at the primary surgical procedure and
dialy during the stay in the hospital ( 5mL venous blood per day).
4. In patients with a fracture of the femur, a small muscle
biopsy will be obtained in the fracture region.
5. For all patients bone healing will be followed closely during standard
follow-up. The development of delayed-union/ non-union or re-fracture in the
primary group and persistence of the non-union in the non-union group will be
noted.
6. Blood samples will be collected during standard follow-up at several time
points.
These time points are, approximately:
- 4 weeks after the primary surgical procedure
- 3months after the initial fracture
- 6 months after the initial fracture
7. During secondary surgical procedures:
A. Patient with a non-union fracture of the femur or tibia who will undergo
surgical correction, or
B. Patient undergoing removal of the intramedullary device after healing of the
fracture of the femur or tibia.

Only a venapuncture will be done during this study, which will not include
extra risk or a high extent of the burden associated with participation.
In patients with a femur fracture, a muscle biopsy will be obtained during the
surgical procedure. A small risk of haematoma and bleeding is existent.