Keywords
bone grafting - bone xenograft - experimental animal model - fracture healing - hematology
Introduction
Blood is a reflector of the status of any animal and has thus been used to know physiologic
and pathologic states and for both diagnostic and prognostic evaluations of various
conditions in animals.[1]
[2]
[3]
[4] Hematology is, therefore, performed to investigate metabolites in the body, responses
to exposure to antigens, and the state of diseases in animals as different pathologic
conditions or exposure to certain conditions affect some specific blood parameters.[1]
[5]
[6]
[7]
In the use of hemogram for diagnosis in rabbits, care must be taken as the hematological
parameters are affected by so many factors. Age, gender, breed, ambient temperature,
diurnal rhythm, and even mere transportation have been reported to cause derailment
in the hemogram of rabbits.[8] For example, lymphocytopenia, leukocytosis and increased packed cell volume (PCV)
have been reported in rabbits transported at 28°C for up to 3 hours, while cold stress
was also reported to increase red blood cell (RBC) count. Furthermore, white blood
cell count (WBC) varies due to diurnal fluctuations and variation, with total WBC
being observed to be at its lowest in the later afternoon and evening when compared
with the earlier hours of the day.[8] In addition, the erythrocytes also vary with gender, as the male rabbits have been
observed to have a slightly higher erythrocyte count than females.[8] Also, infectious diseases do not typically cause leukocytosis in rabbits but present
a shift from lymphocyte-predominant to neutrophil-predominant differential counts;
furthermore, acute infections can be characterized by leukopenia with normal differential
count.[8] Anemia, neutrophilia, leukocytosis, and monocytosis were reported in hepatic coccidiosis
rabbit model contrasting with the eosinophilia known to accompany parasitic diseases
in other animals.[8]
In fracture healing, monitoring of hematological parameters play a vital role as any
deviation from the physiological reference range could be an indication of infection
or graft response and, therefore, require urgent attention to avert the possibility
of mal-union, delayed union, or non-union.[9] Hematology is, however, characterized by fluctuation within the normal physiological
ranges, which may make its use in diagnosis difficult.[9] Decrease in total RBC is seen following trauma and surgery.[9] Stress, trauma, and surgery are known to cause leukocytosis, lymphocytopenia, and
neutrophilia, which could also fluctuate within normal ranges after the initial problem
subsides. The increase or decrease of basophils, monocytes, and eosinophils could
be subjective in fracture healing monitoring.[9] Due to the scarcity of information on hematological response of fracture healing
in animal models, we, therefore, report in the present study, the observed hematological
changes in rabbits that underwent xenografting of caprine demineralized bone matrix
(CDBM).
Materials and Methods
Experimental Design
The present study was approved by the animal research ethics committee of the institution
under the number (UDUS/FAREC/2019/AUP-R0–5). Twenty-four (24) male rabbits (2.5 ± 0.5kg)
were randomly grouped into 3 groups, with 8 rabbits in each group. The groups were
based on the treatment they received: autologous bone graft group (ABG), unfilled
negative control group (NC), and CDBM group.
Preparation of CDBM and Critical Bone Defect
The preparation of the DBM, creation of the defect on the ulna, and bone grafting
were performed with modifications following Arpağ et al.,[10] Monazzah et al.,[11] and Bigham-Sadegh and Oryan.[12]
Blood Sample Collection and Hematological Analysis
Blood samples were collected presurgery to serve as the baseline data for the hemogram,
with specific interest in complete blood count (CBC) and leucocytic differential count.
The blood samples were collected through cardiac puncture under xylazine-ketamine
anesthesia. The samples were stored in a ethylenediaminetetraacetic acid (EDTA) sample
bottle (JRZ Plastilab, Beirut, Lebanon) and processed under two hours of collection
as adopted by Chineke et al.[7] The blood samples were subsequently collected on the 28th and on the 56th postoperative days for evaluation. This was with modification from Ajai et al.,[13] Bigham-Sadegh et al.[14] and Korkmaz et al.[15]
Data Analysis
The data generated was analyzed using a repeated measure mixed model approach with
two-way analysis of variance (ANOVA) to detect differences in interaction of days
and groups concurrently, and significance level was determined at p < 0.05 using InVivoStat 4.0.2 (Chelmsford, Essex, UK).
Results
The surgery and grafts implantation were performed successfully, and the animals recovered
from anesthesia uneventfully. There was an overall significant leukocytosis (p = 0.0043) on days 28 and 56 compared with the baseline. On day 56, a significant
difference was also observed in the neutrophil count (p < 0.0001), while on day 28, significant neutrophilia was observed in the CDBM group
when compared with day 0; the number of neutrophils had reduced by day 56 though it
was not statistically different from its baseline value. There was significant monocytosis
in the ABG and CDBM groups on day 28, which returned to near baseline by day 56. Although
there was no overall statistical difference in the lymphocyte count (p = 0.4923), on day 28, the lymphocyte counts of the ABG and CDBM groups were significantly
higher from their baseline values. Significantly moderate eosinophilia was observed
in the NC and CDBM groups on day 56. An overall significant difference was also observed
for RBC (p = 0.003), hemoglobin concentration (p < 0.0001), and packed cell volume (p < 0.0001) across the days and the treatment groups. However, there was no overall
significant difference in lymphocyte count (p = 0.4923), basophil count (p = 0.4183), and eosinophil count (0.4806). The result is presented in [Table 1] showing the significant differences across the days and among the groups.
Table 1
|
Day 0
|
Day 28
|
Day 56
|
|
ABG
|
NC
|
CDBM
|
ABG
|
NC
|
CDBM
|
ABG
|
NC
|
CDBM
|
|
WBC X 109 /L
|
3.71 ± 0.43
|
3.76 ± 0.36
|
4.53 ± 0.37
|
4.96 ± 0.36[a]
|
4.90 ± 0.72
|
6.75 ± 0.36*[a]
|
5.51 ± 0.34[b]
|
3.81 ± 0.19*
|
4.11 ± 0.24*
|
|
Neut[a] X 109 /L
|
0.76 ± 0.09
|
0.68 ± 0.08
|
0.81 ± 0.09
|
1.00 ± 0.07
|
0.84 ± 0.11
|
1.30 ± 0.11[a]
|
3.09 ± 0.27[a]
|
1.11 ± 0.13*[a]
|
1.19 ± 0.13*
|
|
Lym[c]bX 109 /L
|
2.90 ± 0.34
|
3.01 ± 0.28
|
3.63 ± 0.29
|
3.82 ± 0.29[a]
|
3.98 ± 0.61
|
5.24 ± 0.26*[a]
|
2.30 ± 0.09
|
2.55 ± 0.19
|
2.73 ± 0.14
|
|
Mono[c] X 109 /L
|
0.04 ± 0.01
|
0.07 ± 0.02
|
0.09 ± 0.02*
|
0.13 ± 0.02[a]
|
0.09 ± 0.01
|
0.21 ± 0.03[a]
|
0.07 ± 0.01
|
0.12 ± 0.02
|
0.12 ± 0.02
|
|
Eosin[d] X 109 /L
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.02 ± 0.01
|
0.03 ± 0.01
|
0.03 ± 0.01[a]
|
0.03 ± 0.01[a]
|
|
Baso[e] X 109 /L
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.01 ± 0.01
|
|
RBC X 1012 /L
|
3.47 ± 0.34
|
3.55 ± 0.12
|
3.58 ± 0.30
|
3.10 ± 0.18
|
3.71 ± 0.31[a]
|
4.51 ± 0.27*[a]
|
3.49 ± 0.27
|
4.97 ± 0.23*[a]
|
3.92 ± 0.21
|
|
Hg[f] (g/dL)
|
9.46 ± 0.46
|
14.09 ± 0.35*
|
11.23 ± 0.37*
|
8.37 ± 0.46
|
9.67 ± 0.47[a]
|
10.35 ± 0.80
|
10.43 ± 0.79
|
10.11 ± 0.39[b]
|
11.55 ± 0.41
|
|
PCV (%)
|
26.00 ± 1.34
|
35.58 ± 1.24*
|
31.50 ± 1.32*
|
24.63 ± 0.57
|
23.63 ± 0.96[a]
|
28.50 ± 0.87*
|
29.63 ± 0.94[a]
|
27.63 ± 0.71[b]
|
31.38 ± 1.19
|
Discussion
Since blood is a reflector of the health status of animals, responses to exposure
to foreign bodies, and the state of disease in animals,[1]
[2]
[3]
[4]
[5]
[6]
[7] we used hematology to evaluate the responses of the experimental rabbits to the
grafted caprine DBM, which is expected to initiate some antigenicity. Several reports
have documented normal hematological parameters in rabbits in different geographical
zones and conditions,[4]
[7]
[8]
[16] but the fluctuation within the normal physiological ranges make its use in diagnosis
difficult,[9] especially in rabbits, in which gender, diurnal rhythm, breed, ambient temperature,
age, and stress have been reported to affect their haematology.[7]
[8]
[16] It is, therefore, imperative to compare hematological responses with the baseline
in any experimental condition, as seen in other studies.[1]
[17]
[18]
Leukocytosis, which is consistent with the stress of surgery, inflammation, and excruciating
pain associated with fracture and fracture healing,[1]
[9]
[16]
[18]
[19] was observed in the three groups. The leukocytosis, which was exceptionally marked
and significant in the CDBM group on day 28, could be as a result of the immune reaction
to the implanted caprine DBM. The value of the leucocyte count had dropped by day
56 to near baseline value, which is indicative of reduced reaction to the implant.
This was the same pattern of reaction that was observed for neutrophil and lymphocyte.
This is in accordance with earlier reports,[16]
[20] which state that in conditions that triggers immunologic response, there is leukocytosis
with marked lymphocytosis.
Many factors, such as time of the day of blood sampling and stress, are known to affect
monocyte, eosinophil, and basophil counts.[16]
[20] Despite this, monocytosis has been reported to be consistent with chronic inflammation
in rabbits,[16]
[20] and the same was observed for CDBM on day 28 when compared with its baseline. Eosinophilia
and basophilia are markers of allergic and hypersensitive conditions. Mild eosinophilia
here was not observed in this study until day 56 in CDBM group. This could be due
to reduced immunogenicity of demineralized bone by the process of demineralization
as reported.[21]
[22]
The reduced PCV observed on the 28th day in all the groups was as a result of blood loss to surgery. This is in accordance
with the earlier reports[1]
[20] that anemia occurs when there is external hemorrhage. However, the animals in all
the groups have increased PCV values by day 56, though the values were not up to their
corresponding baselines. This showed that there was regenerative response to the blood
loss. In the same vein, the hemoglobin concentration also decreased on day 28 and
had risen to almost above the baseline by day 56. On the contrary, the RBC count increased
on days 28 and 56 for all the groups, except for the ABG on day 28, which was lower
when compared with the baseline but not statistically significant. The different patterns
observed for the RBC could be a result of the time of sampling and ambient temperature
that affect the experimental animals.[16]
[20]
The inability to monitor the diurnal rhythm and environmental temperature in relation
to the variation of hematological values obtained are the limitations of this study.
Conclusion
Despite several fluctuations that mar the interpretability of hematology in rabbits.
It can be concluded from this study, the rabbits responded to the CDBM with marked
leukocytosis with neutrophilia, lymphocytosis and monocytosis by day 28, which became
near normal by day 56 post-grafting. This could therefore form a hematological basis
with which the state of bone grafting animal models could be monitored. It is however
strongly recommended that baseline is taken in every study involving hematology in
rabbits, putting diurnal rhythm and environmental temperature into consideration.
