International Journal of Novel Research in Life Sciences Vol.

15 Issue 6

ASSESSMENT OF SELECTED BONE TURNOVER MARKERS IN HEALING TIBIAL FACTURES

1
Owamagbe, E. M., 2Lawson S.D.
1
Department of Chemical Pathology, College of Medical Sciences, Rivers State University
2
Department of Medical Microbiology & Parasitology Faculty of Basic Clinical Sciences, College of Medical
Sciences, Rivers State University

ABSTRACT

Imaging techniques are the standard method for assessment of fracture healing processes. However, these methods
are perhaps not entirely reliable for early detection of complications, the most frequent of these being delayed union
and non-union. A prompt diagnosis of such disorders could prevent prolonged patient distress and disability. Enzyme
linked Immunosorbent Assay (ELISA) was used to carry out a serum assessment of Bone Turnover Markers (P1NP;
Procollagen type 1 amino terminal propeptide, TRAcP; Tartrate-resistant acid phosphatase) was carried out on 60
participants with Tibial Fractures at 24 hours, 6 weeks and 12 weeks interval to assess the progression of bone healing.
Analysis of Variance showed a significant significant increase in serum PINP from 417.5±122.2pg/mL 24 hours after
the fracture to 501.0±146.7pg/mL 6 weeks later and 626.2±183.3pg/mL 12 weeks later (p= 0.0001). Similarly, the
was a significant increase in the average TRAcP from 38.85±12.27 pg/mL 24 hours after the fracture to 58.35±18.36
pg/mL and reduced to 46.68±14.69 pg/mL 12 weeks later (p= 0.0001). The study shows that an increase in serum in
the bone turn over markers corresponds with the healing of the tibial fracture and could be an indicative marker for
the progression of bone healing.

Keywords: bone formation markers, bone resorption markers, TRAcP, P1NP, Fractures

1.0 INTRODUCTION

A fracture is the separation of an object or material into two or more pieces under the action of stress while the fracture
of a bone usually occurs due to the development of certain displacement/ discontinuity in the surfaces within the
bone(1–3). Bone turnover markers are substances that are produced by cells found in the bone(4–6). They are either
direct or indirect products of osteoblasts (formation) or osteoclasts (resorption)(6,7). Examples of bone turnover
makers include, Bone alkaline phosphatase (BALP), Tartrate- resistant acid phosphatase (TRAcP), Procollagen type
I N- Terminal Propeptides (PINP)(8–10). These bone turnover markers reflect the biological synthetic activity of
bone-forming cells (bone formation markers) and the biological destructive activity of bone resorptive cells (bone
resorptive markers);they are sensitive and useful indicators of bone metabolism in bone fracture healing(9–11).
Changes in the rate of bone turnover are an important determinant of bone conditions and therefore, measurements
that correlate with the rate of turnover provide important information in assessing patients with bone conditions(12–
15). In recent times, the study of bone turnover markers has developed greatly. Different biochemical markers are
currently available which make for specific and sensitive appraisal of the rate at which bone is formed and broken
down(16–19).

Bone remodeling is essential in maintaining good bone health through the process of bone formation and resorption
or degradation(20,21). Bone fractures undergoes the process of bone healing and this healing process involves the
following- inflammation, formation of soft callus, hard callus and bone remodeling(22,23). However, failure of the
healing process of a bone fracture may cause delayed union, nonunion and malunion(12,13). The bone turnover
markers are usually released early in bone healing and as such could be used as early markers for bone healing and
also for early detection of the complications of bone healing(14,15,24). It is known that the radiological means of
assessing bone healing in the early stages may be unreliable and also does not provide adequate information till late
in the treatment, which may require the surgeons to assess patients healing process late in the treatment in other to get
a better assessment(4,13,22). An assessment of the bone turnover makers (BTM) will aid in providing additional
information on the process of fracture healing and reduce the need for surgical interventions to assess the status of

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bone healing. Based on the findings of the findings of the study, it is expected that assessment of bone turnover markers
will aid radiological assessment of fracture and improve healing time for fractures. The study was carried out to assess
the relationship between serum Bone turnover markers type-I collagen N-terminal propeptide (PINP) and Tartrate-
Resistant Acid Phosphatase (TRAcP).

2.0 METHODS

2.1 Study Setting

This study was carried out in the accident and emergence (A/E) unit, orthopaedic ward and orthopaedic clinic of the
University of Port Harcourt Teaching Hospital (UPTH), Port Harcourt, Rivers State. The orthopaedic unit serves as
primary provider of care to both staff of the hospital as well as other members of the public.

2.2 Study Population

The study population consists of patients with simple tibial fractures from Road Traffic Accident (RTA) admitted
through the Accident and Emergency (A/E) department, in the orthopaedic ward and those attending the Orthopaedic
clinic of the UPTH will be recruited as participants while the participants immediately after fracture was used as
control subjects.

2.3 Sample Size

The sample size for the study is determined from the formula(12). n = Z2pq/ d2

Where; n = sample size, Z = 95% confidence interval= 1.96, P = proportion of the target population used=4.1%(14)

q = 1.0 – p = 1.0 - 0.041 = 0.959, d = degree of accuracy desired (usually set at 0.05)

n = {(1.96)2 (0.041) (1.0 - 0.959)}/0.025

= {3.841 × 0.041× 0.959}/0.0025 =60

2.4 Specimen Collection and Analysis

A total of 6ml venous blood was collected from each participant under aseptic condition from the ante-cubital vein or
veins of the dorsum of the hand early each morning (8am to 10am) and a part of it was placed in a plain container for
the analysis of serum levels of the Bone turnover markers. Commercial Enzyme-linked immunosorbent assays was
used to determine the levels of the bone turn over markers according to the manufacturer’s instruction(5).

2.5 Ethical Consideration

Approval for the study had been sought from the Ethical Committee of the University of Port Harcourt Teaching
Hospital, before commencement of the study. Departmental approval from the Head of Orthopaedic Department was
obtained. Informed consent was also sought from each study participant recruited in accordance with ethical principles
for the guidance of physicians in medical research (confidentiality, beneficence of participants, non-maleficence to
the participants, and the right to decline or withdraw from the study without loss of benefits).
2.6 Data Analysis

Data obtained from the study was analyzed using the Statistical Package for Social Science (SPSS version 20). Results
was expressed as Mean ± standard deviation. The unpaired t-test was used to compare the mean levels of bone turnover
markers measured at the time of fracture, six weeks after fracture and when the simple properly healed tibial fracture
has occurred from x-ray. The participants were used as their controls. All tests were done at a 95% confidence interval
and a p-value less than 0.05 was considered significant.

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3.0 Results

The above tables show the demographic distribution of the subjects that were recruited into the study. There were 27
(45.0%) male and 33 (55.0%) females. The distribution of the age groups showed that 38 (63.3) of the subjects were
between 20 – 29 years and 22 (36.7%) of the subjects were between 30 – 39 years old.

Table 1: Sociodemographic Data

Frequency (n=60) Percent

Gender
Male 27 45.0

Female 33 55.0

Age groups
20 – 29 years 38 63.3

30 – 39 years 22 36.7

Mean age ±S.D 29.17 ±5.6

S.D: Standard deviation

Table 2 shows significant increase in serum PINP from 417.5±122.2pg/mL 24 hours after the fracture to
501.0±146.7pg/mL 6 weeks later and 626.2±183.3pg/mL 12 weeks later (p= 0.0001). Similarly, the was a significant
increase in the average TRAcP from 38.85±12.27 pg/mL 24 hours after the fracture to 58.35±18.36 pg/mL and reduced to
46.68±14.69 pg/mL 12 weeks later (p= 0.0001).

Table 2: Comparison of BTM at 24hrs, 6weeks and ≥12weeks

Variables
24hrs 6weeks ≥12weeks p-value

PINP (pg/mL) 417.5±122.2 501.0±146.7 626.2±183.3 0.0001*

TRAcP (pg/mL) 38.85±12.27 58.35±18.36 46.68±14.69 0.0001*

P1NP; Procollagen type 1 amino terminal propeptide, TRAcP; Tartrate-resistant acid phosphatase. All data are
presented in Mean ±standard deviation, *difference is statistically significant (p < 0.05).

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Table 3 indicated that there was an increase in PINP corresponds with an increase in TRACP at 24 hours, 6 weeks
and ≥12weeks.

Correlation of BTM at 24hrs, 6weeks and ≥12weeks

24hrs 6 weeks r ≥12weeks r
r (p-value) (p-value) (p-value)
PINP v TRACP 0.56 (0.0001) 0.56 (0.0001) 0.56 (0.0001)

There was a positive correlation between all BTM at 24 hours, 6weeks and ≥12weeks. All
correlations were statistically significant (p<0.05)

P1NP; Procollagen type 1 amino terminal propeptide, TRAcP; Tartrate-resistant acid phosphatase

4.0 Discussion

The serum level of PINP is highest in those with fracture of ≥ 12weeks duration, followed by those participants with
fracture of 6weeks and least in the control participants. The study showed a progressive increase in the mean serum
PINP levels at 24 hours till ≥12 weeks as the healing process moves from no callus through soft callus formation to
bony callus formation. The results showed significantly higher levels of PINP at ≥12 weeks, where there was
radiological and clinical evidence of bone healing. This finding is consistent with the reports of other studies which
showed elevated levels of PINP as an indicator of osteoblastic activities after a fracture(25,26). The elevated
expression of PINP has also be reported to be associated with the localized callus formation at the location of a bone
fracture, which is an indicator of progressive bone healing. However, the difference in PINP level at 6 weeks was not
statistically significant compared to the PINP levels at ≥12 weeks among the subjects, this is the period of the
conversion of the soft callus to the bony callus which involves the mineralization(27,28). Consequently, this is an
indication that soft callus formation is taking place at 6weeks in most of the individuals with fracture.
The mean plasma level of TRAcP is highest in those with fracture of 6weeks duration, followed by those participants
with fracture of ≥12weeks and least in the control participants. The analysis of variance (ANOVA) shows that the
difference of mean plasma TRAcP levels across the groups is statistically significant (p ˂ 0.0001). There was a
consistent elevation in mean plasma TRAcP level at 24 hours to 6 weeks of the fracture among the subjects which are
at the point of maximum osteoclastic activity to make room for the deposition of new bone and the removal of the
dead bone. However, there was a relative decline in mean plasma TRAcP levels at the 12 th week of the fracture when
compared to the mean plasma TRAcP level at 6th week, this is due to less osteoclastic activity. This finding is
consistent with the reports of similar studies(29,30).
Bone fractures stimulate an increase in the number and activity of bone metabolic units of osteoblasts and osteoclasts,
which result in an increase in the osteoclastic activity (resorption) at the early stage of bone fracture healing
subsequently removing necrotic tissues at the fracture margins. This event is associated with an equivalent decrease
in bone-formation markers, which is associated with an inhibition of osteoblastic synthetic activity(1,29,30). It is
caused by the release of cytokines from inflammatory cells(2,3,31,32). The increase in the resorption biomarkers may
be caused by the traumatic process itself, or be associated with immobility since this stimulates resorption and inhibits
bone formation(25–27). It has been reported that there were lower levels of tartrate-resistant acid phosphatase5b
(TRAcP 5b) in patients who developed non-unions compared to patients with normal healing. TRAcP 5b is a direct
marker of osteoclastic activity and bone resorption, while BsALP and PINP are an indirect measure of osteoblastic
activity by reflecting on collagen deposition and degradation(28–30). Following a bone fracture, increased bone
metabolism is observed in patients with normal healing. As shown in the current study, this was reflected by a sudden
increase in osteoclastic markers such as TRACP 5b, during the first few weeks as previously reported(1–3,31).
Moghaddam et al assessed the role of TRAcP 5b as a prognostic indicator of fracture healing and they observed lower
serum TRAcP 5b activity at early stages in patients with delayed fracture healing than in patients who had presented

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with normal fracture healing, perhaps reflecting disturbances in bone resorption during the normal process of bone
regeneration(25–27). This is in line with the findings of the current study which showed a significantly elevated level
of serum TRAcP in subjects with bone fracture undergoing a normal healing process. Levels of TRAcP 5b peak seven
days after osteosynthesis in ankle fractures and after two weeks in tibial fractures, remaining high at 24 weeks(1,2).
Similarly, it has been reported that a higher increase in TRAcP activity in fractures probably reflect the size of the
fractured area and the need for more extensive bone remodeling.
5.0 Conclusion

The findings of the study showed a marked increase in the serum levels of bone resorption marker as fracture healing
occurs in the subjects. The elevated levels of this bone turnover marker corresponded with radiological observations
of callus formation from the 6th week of the fractures among the subjects, which indicated a progression in bone
healing. Consequently, the observed elevation of these bone turnover markers was indicative of the extent of bone
resorption and bone turnover activities which could be used as early markers in the assessment of the healing process
in bone fractures.

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