Hematological Changes after Caprine Demineralized Bone Matrix Implantation in Ulnar Critical Defect of Rabbit Model

Abstract

Objective  Several animal models have been used in fracture healing and bone graft studies, but hematological responses are seldom reported. Therefore, the present study reported the hematological changes observed in rabbits that underwent xenografting of caprine demineralized bone matrix (CDBM).

Method  Twenty-four (24) male rabbits (2.5 ± 0.5kg) were acquired for the purpose of this study and were randomly assigned to three groups: autologous bone graft (ABG), unfilled (NC), and caprine demineralized bone matrix (CDBM). Blood samples were collected through cardiac puncture under xylazine-ketamine anesthesia on day 0 (baseline), and on days 28 and 56 postsurgery and were analyzed manually within 2 hours of collection. Statistical analysis was performed using a two-way analysis of variance (ANOVA) with repeated measures, and a p -value < 0.05 was considered significant.

Result  There was an overall significant difference in the values of total white blood cell count ( p  = 0.0043), neutrophil count ( p  < 0.0001), monocyte count ( p  = 0.0184), red blood cell count ( p  = 0.003), hemoglobin concentration ( p  < 0.0001) and packed cell volume ( p  < 0.0001) across the days and the treatment groups. There was, however, no overall significant difference in lymphocyte count ( p  = 0.4923), basophil count ( p  = 0.4183), and eosinophil count (0.4806) within days.

Conclusion  Response to CDBM grafting in rabbits could, therefore, be said to be characterized by marked leukocytosis with neutrophilia, lymphocytosis, and monocytosis by day 28 of postgrafting. This could form the basis with which hematology can be used to monitor body response of bone graft animal models.

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 28 ^th and on the 56 ^th 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. Hematological results (mean ± SEM) of experimental animals presurgery (day 0), on day 28 and on day 56 ( n  = 8) .

	Day 0			Day 28			Day 56
	ABG	NC	CDBM	ABG	NC	CDBM	ABG	NC	CDBM
WBC X 10 9 /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 10 9 /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 10 9 /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 10 9 /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 10 9 /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 10 9 /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 10 12 /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

Abbreviations: ABG, autologous bone graft; CDBM, caprine demineralized bone matrix; NC, negative control; PCV, packed cell volume; RBC, red blood cell count; SEM, standard error of the mean; WBC, white blood cell count.

Different alphabets indicate that the means differ significantly ( p  < 0.05) from day 0 within the same group, while the means bearing an asterisk differ significantly ( p  < 0.05) from ABG for their respective days.

^aNeutrophils.

^bLymphocytes.

^cMonocytes.

^dEosinophils.

^eBasophils.

^fHemoglobin concentration.

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 28 ^th 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.