Experimental Study on the Early Immunological Rejection in

Xenotrans

lantation of Rabbit Fracture Hematoma Cells

Yan Fang

, Xia Dong

, Zhengjin Liu

and Xiaoping Ying

Laboratory of Molecular Pathology, Shaanxi University of Chinese Medicine,

（

Xianyang 712046

）

;

Zhongshan Hospital Xiamen University,

（

Xiamen 361004

）

Keywords: Xenotrans plantation, hematoma cells, rejection.

Abstract: Objective: To investigate if xenotransplantation of rabbit fracture hematoma cells in the broken ends of

fractured bone can lead to obvious hyper acute and acute immune rejection. Methods: We conducted a trial

involving 90 rabbits with 4~6 weeks old, weighting 2.2~2.6kg, male and female in half. They were

randomly divided into three groups: fracture group (n=30), fracture transplantation normal saline group

(n=30), and fracture transplantation hematoma group (n=30).3 days after animal models of sawed-off

cubital bone was established, rabbits fracture hematoma cells which were derived from the ordinary livor

blue rabbits’ fracture hematoma and cultured in vitro were transplanted into the broken ends of fractured

bone. Ten rabbits were sacrificed in each group at the 1st, 4th, and 8th day after the transplantation.

Immunohisto chemical method was adopted to observe the survival rate of fracture common chinchilla

rabbit fracture hematoma cells, CD68+ macrophage infiltration and splenic lymph follicles. Immunohisto

chemical SABC method was used to detect the expression of CD68 in the macrophages of New Zealand

white rabbits around the allograft. Results: After transplantation, the fracture hematoma cells of xenogeneic

rabbits survived a lot at the fracture end, and the cells’ structure was normal. The splenic lymphoid follicles

did not proliferate significantly in each group and there was no significant difference in the expression of

CD68 macrophages at each interval between groups. Conclusion: There was no significant immunological

rejection in the early stage of xenotransplantation of rabbit fracture hematoma cells in the broken ends of

fracturedbone.

1 INTRODUCTION

It is well known that the formation of the hematoma

after fracture can promote fracture healing. and it

has also been reported that there are cases and

experiments in fracture treatment with hematoma

[1,2] and these results implied that after

transplantation of the fracture hematoma cells, the

subjects showed early callus formation, large

amount callus formation, and the average fracture

healing time was significantly shorter, indicating

that the fracture hematoma had a significant effect in

fracture healing. However, these studies are only

applicable to autologous hematoma cells, while

limited sources of autologous hematoma cells

cultured in vitro proliferation and poor factors limits

its practical application in the clinic. Therefore,

heterologous transplantation of hematoma cells

could be the better choice. Although the fracture

hematoma cells are in a primitive state with weak

auto antigenicity, there are no reports about whether

the heterogeneous hematomas cell transplantation

will lead to significant immune rejection. Fracture

hematoma cells of heterologous rabbits will be

transplanted into the fracture model in this

experiment to observe the early immune rejection,

and explore the osteogenic potential of heterologous

hematoma cell transplantation in fracture healing.

2 MATERIALS AND METHODS

2.1 Materials

2.1.1 Animals and Groups

The experimental animals were New Zealand white

rabbits bought from the experimental animal center

of The Fourth Military Medical University. We

conducted an experiment involving 90 rabbits with

4~6 weeks old, weighting 2.2~2.6kg, male and

female in half. They were randomly divided into

three groups: fracture group (n=30), fracture

transplantation normal saline group (n=30), and

fracture transplantation hematoma group (n=30).

2.1.2 Cell Culture Reagents and Antibodies

DMEM medium (American Hyclone company),,

which include 100IU/ml penicillin, 100 g/ml

streptomycin (Shanghai Biological Engineering

Technology Co Ltd), 15% newborn bovine serum

(PAA); BrdU and anti BrdU antibody, Goat anti-

rabbit CD68, IgM and IgG first antibody, Rabbit

anti-goat second antibody (Fujian Maixin biological

Technology Development Company).

2.1.3 Main Instruments

OLYMPUS IX70-SIF2 inverted microscope, table

model high speed centrifuge (BIOFUGE STRATOS,

Heraeus company).

3 METHODS

3.1 Isolation and Culture of Fracture

Hematoma Cells

Three days after the model of common livor blue

rabbit femoral fracture, the rabbits were anesthetized

with 3% pentobarbital sodium (30mg/kg) through

ear vein. Hematoma cells from fracture sites were

extracted under aseptic condition, and were put into

Heparin Sodium Single-use Automatic Quantitative

Tube for Blood Specimen Collection (purchased

from Wuhan Zhiyuan Medical Technology Co., Ltd)

, 3ml each. The specimen was shaken up repeatedly

to avoid the formation of small clots, moved to

super-clean worktable and added with 2ml DMEM.

After consecutive pumps with No.4 syringe needle,

it was made into single cell suspension. Following

centrifuge(1000r/min, 10min) to get rid of fat and

supernatant, the remaining cell components were

inoculated into 50ml culture bottle with DMEM,

2ml each. After 7 days under standard environment

(37C°, saturated humidity,5% CO2), the culture

medium was totally replaced and the suspending

hemopoietic stem cells as well as unattached cells

were removed. Then culture medium was replaced

every 3～4 days, and cell shape and growth state

were observed daily through inversion microscope.

Subculture: when a complete layer formed, the cells

were rinsed three times with PBS (purchased from

Hyclone) with the supernatant removed and were

treated with 0.25% trypsinase (from Gibco) and

0.02% EDTA (from Gibco) for 5 min at the ratio of

1∶2 before subculture.

3.2 BrdU Labeling of Fracture

Hematoma Cells

When formed a complete single layer, these 2nd

generation hematoma cells were incubated with

BrdU (terminal concentration:10 mmol/L) for 24h,

followed by washing with non-serum DMEM

medium for 5 times, and were treated with 0.125%

trypsinase and 0.01% EDTA before they were made

into 1×108/ml cell suspension for cell

transplantation [3-4].

3.3 Rabbit Fracture Model

With ulna exposure, the New Zealand rabbits were

conventionally anesthetized, fixed under aseptic

conditions, Ulnas were sawed cross-sectionally

across the middle part with sterile hacksaw blade,

then muscle and skin were sutured. Skin was

disinfected and bound with gauze. 1~5 days after

operation, rabbits were administered penicillin

intramuscularly every day (0.3million unit/kg)

3.4 Transplantation of Fracture

Hematoma Cells

Hematoma cells were transplanted on the 3rd day of

fracture modelling[5]. Group for fracture

transplantation hematoma cell: BrdU marked

hematoma cells were injected with needles

perpendicular to ulna and fracture cross-sections,

with 10mm and13mm in depth, inoculated with 5μL

of cell suspension, and then withdrawn needle

slowly after 10min. Saline group of fracture

transplantation: The physiological saline was

injected according to the above method.

3.5 Test Results

3.5.1 The Growth of Hematoma Cells

After heterologous transplantation, 10 rabbits from

each group were executed at the 1st, 4th and 8th

days, respectively. Their ulnas were taken out and

decalcificated for 60d with 15% neutral

EDTA(purchased from Gibco), continued with

dehydration in a graded series of alcohol before they

were made into paraffin-embedded sections. DAB

staining with anti-BrdU monoclonal antibody ABC

method witnessed that BrdU positive reactants

located at nucleus were brown, granular-like or

distributed diffusedly. 5 sections from each New

Zealand white rabbit were selected and observed

under low-magnification microscope (×10) to count

the total number of Brdu positive cells for statistical

processing.

3.5.2 The Infiltration of Neutrophils at the

Broken End of the Fracture on the

First Day After Xenotransplantation

Was Observed Under Light

Microscope

5 pieces of the ulna paraffin sections of New

Zealand white rabbits executed on the first day after

operation were selected and stained with

conventional HE. 10 slices of vision were taken

from each slice, and neutrophils were counted at

high power microscope (×40). Then the neutrophils

were counted and processed statistically.

3.5.3 Immunohistochemical S-P Assay Was

Used to Detect CD68 Positive

Mononuclear Macrophages at

Fractured End

Immunohistochemistry S-P kits and CD68, produced

in Zgmed, were purchased from Fujian Maixin Bio-

tech Co.Ltd. CD68 positive sections were chosen as

positive control, while PBS, in place of first-

antibody, was taken as negative control. Positive

reaction arose when the total cell count of brown

granules was more than 10%, and scoring was done

based on positive cell count percentage and coloring

intensity, and the sum of the two indexes were

analyzed statistical.

3.5.4 Count Lymphoid Cells

On the 1st, 4th and 8th day after heterologous

transplantation, 10 rabbits from each group were

executed. Their ulnas and spleens were taken out

and rinsed with saline, fixed with 4% neutral

formaldehyde solution and dehydrated in a graded

series of alcohol before they were made into

paraffin-embedded sections. After routine HE

staining, the largest section was observed from five

randomly selected views under four-fold objective

lenses. Meanwhile, lymphoid follicle count was

recorded (when lymphoid follicle covered the

middle-line, the left and upward sites rather than the

right and downward sites were recorded). Then the

data were analyzed statistically andχ2 test was

adopted.

3.6 Statistical Methods

All the data were analyzed with SPSS13.0 software.

Measurement data was expressed with mean

±standard deviation(

−

±s), and χ2 test was adopted.

4 RESULTS

1.The 3th day of primary culture (Fig. 1), it can be

seen sporadic hematoma cells, and most of them are

short spindle or triangle. The 12th day of Primary

culture (Figure 2), hematoma cells were fused into

monolayer and primary growth was completed.

Fig. 1: the 3

day of primary culture (×100).

Fig. 2: the 3th day of primary culture (×100).

2. Growth status of hematoma cells at different

times in Hematoma Cells group: On the 1st, 4th and

8th days after heterologous transplantation, many

BrdU positive hematoma cells with normal structure

were visible in the transplanted region, and no

obvious degeneration and necrosis could be found.

Positive cell count results showed that according to

comparison of positive cell counts in different post-

operational periods(

−

±s), BrdU positive cell count

on the 4th day（57.20 ±4.632） outnumbered that

of on the 1st day（38.72 ±5.217） and 8th day（

46.43 ±4.345） (P＜0.05).

3.One day after xenotransplantation in New

Zealand white rabbits, neutrophils counts in the

broken ends of fracture bone (

−

±s): fracture group

(8.6 + 0.72), saline group (9 + 0.55) and hematoma

cell group (8.9 + 0.43), neutrophils counts have no

significant difference in each group (P > 0.05)

(Figure 3).

Figure 3: Neutrophilic granulocyte infiltration around the

broken end of the fracture (1

day) (×100).

4. The expression of IgM and IgG around the

broken end of the fracture bone in New Zealand

white rabbits: no obvious brown granules.

5. Different number of CD68 positive

mononuclear macrophages was found in both the

hematoma group and the control group (Figure 4).

(Table 2)

Table 2: CD68 positive cells counts of the three rabbits’

groups at different time(

−

±s).

N Fracture group Saline group Hematoma cell group

1th day 30 0.8±1.1 0.9±0.5 0.9±0.7

4th day 30 4.6±1.2 4.7±1.0 5.0±0.8

8th day 30 5.0±0.7 5.2±0.8 5.3±0.6

Note: no significant difference between each group on the

1th day, 4th day and 8th day after operation (P > 0.05).

Figure 4: Count of lymphoid follicles in the spleen (4th

day) (×200).

6.The count of lymphoid follicles in the spleen

(Table 1)

Table 1: lymphoid follicles counts of three groups of

rabbits after transplantation at different time(

−

±s).

Fracture group Saline group Hematoma cell group

1st day 17.6±0.52 18.0±0.55 17.9±0.43

4th day 17.5±0.46 17.7±0.55 18.2±0.50

8th day 18.0±0.34 18.2±0.43 18.4±0.35

Note: no significant difference between each group on the

1th day, 4th day and 8th day after operation (P > 0.05).

5 DISCUSSION

It is known that the hematoma formed by fracture

plays an important role in the process of fracture

healing. The studies on extracting hematoma cells to

promote fracture healing are also confirmed that

hematoma cells have a significant role in fracture

healing [2], however, duo to the limited sources of

cell and the time difference of in vitro hematoma

culture, the practical application chances of it are

greatly reduced. The xenogeneic fracture hematoma

cells are widely distributed, which can be stored in

advance, and also highly proliferative and

multipotential, so they can be one of the best seed

cells to replace autologous hematoma cells

transplantation for the treatment of fracture healing

[6]

. In recent years, the proliferation of in vitro cell

culture is increasingly mature, which makes it

possible to proliferate a large number of primary

cells in the short term and can be reserved for a long

time. In theory, it is considered that the fracture

hematoma cell is a relatively primitive cell with

weak antigenicity. Therefore, allograft

transplantation may cause mild or even no immune

rejection. But up to now, no specific experimental

study at home and abroad was reported.

Schuurman et al. [7] divided xenograft rejection

in the early stage into three categories: hyperacute

rejection (HAR), acute humoral xenograft rejection

(AHXR), and acute cellular xenograft rejection

(ACXR). Studies have shown that the graft non-

function were mainly due to the hyperacute rejection

and the acute dissimilar rejection of the body fluid.

Hyperacute rejection is a leading cause which occurs

within 24 hours after transplantation, It is mainly the

antibody mediated mechanism, which is the humoral

immune response caused by the natural antibody

IgM, and the natural antibody IgG also plays a

certain role[8-10]. Dehoux [11] suggests that anti

IgM and IgG play an important role in activating

endothelial cells and complement. Especially the

induction of anti -Gal IgG is significantly elevated in

AHXR, which may play a major role. The diagnostic

criteria for antibody - mediated acute graft rejection

include 3 basic characteristics [12]: (1)

morphological evidence for acute tissue injury. (2)

immunological evidence of antibody action. (3)

Serological evidence of circulating donor specific

human leukocyte antigen (HLA) antibody or other

donor epithelial cells antigen specific antibodies.

One of the characteristics of AHXR is the

infiltration of all kinds of cells to the grafts. The

existence of neutrophils has a certain predictability

in the diagnosis of AR, and it may represent early

immune response is activated [13-15].

Fischbeck[16] study shows that DXR is mediated by

immune cells such as mononuclear cells.

Mononuclear phagocyte system responsible for

recognition and rejection of xenogeneic antigen in

xenotransplantation [17]; The lymphoid follicles in

the spleen increases when the antigen and blood

circulation enters the spleen and causes humoral

immune response [18]. The results showed that after

the ordinary rabbits hematoma cells were

transplanted into New Zealand rabbits fracture of 1

days、 4 days and 8 days after transplantation, a

large number of xenohematomas survived in the

transplanted region, no obvious degeneration and

necrosis were found and no obvious IgM and IgG

deposition was found around the broken end of the

fracture. There was no significant difference in the

infiltration of neutrophils and CD68 positive

macrophages in the fracture area between each

group in different time. At the same time, there was

no significant difference in the number of splenic

lymphoid follicles in and between groups at different

time, and no significant proliferation of the splenic

lymphoid follicles was found. All these indicate that

there is no obvious rejection reaction between

transplanted rabbit xenogeneic hematoma cells and

their receptors in early stage, and good

histocompatibility also imply that allogeneic

hematoma cells transplantation is feasible, which

provides an experimental basis for future treatment

of fractures or bone defects.

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