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Sadat Veterinary Medical Journal

ISSN: 1110-9750

Repair of Bone Gap Defect Using Human Wharton Jelly Derived Stem Cells in

Canine Model: Radiological and Histopathological Study

Ahmed Sharshar1*, Mostafa Abd Elgaber2, Gehan Abd Elfatah3, Anis Anis4

1 Department of Surgery, Anesthesiology and Radiology, 4 Department of Pathology, Faculty of

veterinary medicine, University of Sadat City, Egypt

2 Department of Pathology, Faculty of Veterinary Medicine, 3 Department of Clinical Pathology,

Faculty of Medicine, Menoufia University, Egypt

* Corresponding author: E-mail: ahmed.sharsharvet.usc.edu.eg Accepted: February 2018

Abstract:

This study was conducted to evaluate the ability of human Wharton jelly derived stem cells (WJSCs) to

induce bone formation when implanted in canine tibial gap defect. As well as to insure absence of

immune rejection against stem cells when implanted xenogenically. It was carried out on nine apparently

healthy males of mongrel dogs weighing 20 to 25 kg. The animals were randomly divided into three

equal groups (1, 3 and 6 months) according to the observation periods. Two 10 mm diameter holes were

surgically created at the proximal third of the tibia. The first hole was filed at the time of the operation

with WJSCs suspension while, the second one was left empty as control negative. Radiological and

histopathological studies revealed that holes filed with WJSCs showed new bone formation which was

faster and better compared to control one. As well as, no immune reaction was detected against WJSCs

when implanted xenogenically. Our results support the hypothesis that mesenchymal stem cells can be

used for bone grafting between different species without the fear of immune rejection.

Key words: Human Wharton jelly derived stem cells, Gap defect, Xenogenic, Dog, Histopathology

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INTRODUCTION

Bone is the most transplanted human and animal’s

tissue. When bone injury occurred, it retains a

good capacity to repair itself (Schmidt-Bleek et

al., 2014). Spontaneous healing usually not

allowed in some cases of bone affections. In such

cases, surgical intervention and bone regeneration

strategies are required to induce bone healing

(Jafarian et al., 2008; Moghaddam, et al., 2015;

Westhauser et al., 2015; Stanovici et al., 2016).

Autologous bone grafts still considered as the gold

standard for treatment of bone defects (Salgado et

al., 2004). Although it is superior to allografts and

xenografts (Finkemeier, 2002), its application in

orthopedic felids is usually limited by a

considerable donor site morbidity and the limited

amount that can be supplied by the donor (Spitzer

et al., 2002; Kim et al., 2009). Allografts and

xenografts are the second widely used bone

grafting materials. It possesses many advantages

over autograft bone in terms of; it reduces the

donor morbidity, it can be supplied with large

number and different shapes as well as, it

constitutes a major source of osteogenic cells to

the implantation site (Cho and Lee, 2006,

Stievano et al., 2008; Heo, et al., 2011; Emara

et al., 2013a&b). their clinical application is

usually hindered by the risk of immune rejection

and pathogen transmission to the implantation site

(Shegarfi & Reikeras, 2009; Nandi et al., 2010;

Herberts et al., 2011).

Several biomaterials have been used as bone

substitutes. It well accepted and tolerated by

living body. It also, possesses the advantages of

an unlimited availability, good osteoconductivity

and high biocompatibility (Hak, 2007; Ghosh et

al., 2008; Nandi et al., 2008; Rahaman et al.,

2011; Emara et al., 2013b). On the other hand,

most of the known bone substitutes lakes

osteoinductive properties which made them

unable to heal large bone defects when used alone

(Ajeesh, 2010; Emara et al., 2013 b).

Recently tissue-engineering in the form of

cells capable of osteogenic activity was

introduced into orthopedic fields an alternative to

bone grafting materials for inducing bone

regeneration (Jang et al., 2008; Jafarian et al.,

2008; Chen et al., 2009; Udehiya et al., 2013).

This approach has several advantages over

traditional methods of bone grafting; including

ease of handling and manipulation of the cells,

good quality repair, low morbidity at the donor

site and low risk of immunorejection and

pathogen transmission (Pountos et al., 2007).

Human and animal bodies houses several

types of uncommitted progenitor stem cells

capable of giving rise to daughter cells. It can

grow and differentiate into one or more types

(Schwartz & Reyes, 2002; Smith & Webbon,

2005; Pountos et al., 2006; Jung et al., 2008;

Yamachika & Iida, 2013). They were isolated

from different location in the body (Tuli et al.,

2003; Pountos et al., 2006). Among adult stem

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cells, mesenchymal stem cells (MSCs) are the

most commonly used type suitable for bone tissue

engineering. It represents an ideal stem cell source

for cell therapies due to their ease of isolation,

expansion and their ability to differentiate into

different tissues under certain stimuli (Chao et al.,

2007; Patel et al., 2008; Udehiya et al., 2013;

Brennan et al., 2014).

Autologous and allogenic MSCs were used

alone or seeded with synthetic or natural

osteoconducting matrix for bone regeneration

(Planka et al., 2008; Jafarian et al., 2008; Pagni

et al., 2012; Yamachika & Iida, 2013). The cells

may be implanted directly after harvesting

(Centeno et al., 2006; Le Nail et al., 2014;

Scaglione et al., 2014; Thua et al., 2015;

Ajiboye et al., 2015), implanted after being

isolated, expanded and directed to form specific

tissues using specific stimuli (Dong et al., 2013;

Brennan 2014).

The use of autogenic MSCs has its limitations;

in which to obtain the proposed number of the

cells, a large quantities of bone marrow are

required to be aspirated which subjects the patient

to more than one operation (Hernigou et al.,

2005; Kim et al., 2007). In addition, several

weeks are required for expansion of the cells

before implantation (Watson et al., 2014). The

availability of both allogenic and Xenogenic

MSCs and its ability to overcome host immune

rejection have made these cells an attractive

alternative to autogenic MSCs for reconstructive

surgery (Nishio et al., 2006; Kim et al., 2007;

Jung et al., 2009; Stanovici et al., 2016;

Westhauser et al., 2016).The aim of this study is

to investigate the ability of human Wharton jelly

derived stem cells (WJSCs) to induce new bone

formation when used to reconstruct an artificially

induced critical size bone gap defect in canine

tibia. As well as to

MATERIALS AND METHODS

1. Experimental design

The study protocol followed the guidelines of

faculty of veterinary medicine, University of

Sadat city, Egypt for the use and care of animals.

Nine males apparently healthy, Mongrel dogs

weighing 20 to 25 kg. were used in this study. The

animals were randomly divided into three equal

groups 1, 3 and 6 months according to the

observation periods. The dogs were used as

recipient for WJSCs

2. Isolation, Characterization and processing

of Human Wharton jelly derived stem cells

(WJSCs)

2.1. Isolation and culture of Wharton jelly

derived stem cells (WJSCs)

WJSCs were isolated from donated umbilical cord

(UC) tissue samples (n=15). Umbilical cord

samples were collected from normal, full-term

deliveries following cesarean section. Informed

consent from all others was taken. Umbilical

cords were transported to the laboratory in normal

saline and cell isolation was carried out within

24 h. from tissue collection. After removal of

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arteries and veins, Wharton jelly was cut into 0.5-

to 1-cm3 pieces and suspended in fresh complete

nutrients medium which includes: Dulbecco

modified Eagle low-glucose media (DMEM-LG)

with L-glutamine, 10% FBS, Penicillin-

streptomycin (10,000 U/ml and 10,000 µg/ml),

Fungizone (250 µg/ml), all from Lonza. The

flasks were incubated in a 37°C humidified

incubator with 5% CO2 to allow cells to migrate

from the explants. The first change of the media

was at day 7 then the media was changed twice

weekly until reaching 70% to 90% confluence by

inverted microscope (Huang et al.,2010).

2.2.Characterization of MSCs by

flowcytometry

The harvested MSCs were characterized by

flowcytometric analysis for PE CD34

(Imunostep), PE CD44 (BD Pharminogen) and

FITC Oct3/4 (BD Pharminogen). 100 ul of cell

suspension was incubated with 10 ul monoclonal

antibody incubated in the dark at 4 c for 20

minutes. The tubes were washed with 2 ml

phosphate buffered saline (PBS) at 1800 rpm for

5 minutes. Analysis was performed using the

BECTON DICKINSON software (BD

Biosciences).

2.3. WJSCs processing for application:

After 80% confluence was reached, and at least 3-

4 hours before the operation the adherent cells

were harvested by trypsin-EDTA 0.25% solution.

Viability was assessed by trypan blue 0. 4 %

(Sigma). Cells was counted by hemocytometer

and 20 × 106 cell suspension in DMEM-LG were

stored in Co2 till the operation time.

2.4. Surgical procedures

2.4.1. Anaesthesia and Preoperative animal

preparation technique:

All animals were pre-medicated with I/V injection

of mixture of atropine sulfate 0.05 mg/kg

(Atropine sulfate®: 1mg/ml Med. Co., ARE) and

diazepam 1 mg/kg (Neuril®: 0.5% sol. Memphis

Co. for Pharm. &Animal Ind. Cairo A.R.E).

Anaesthesia was induced immediately through

I/V injection of a mixture of Ketamine 10 mg/kg

(Ketalar®: 5% sol. Amoun Co. A.R.E.), and

Xylazine 1 mg/kg (Xylaject®: 2% sol. ADWIA

Co., A.R.E). The anaesthetic depth was

maintained with 2.5 % thiopental sodium

(Thiopental®: EPICO Co., ARE), administrated

by I/V rout (Schmidt et al., 1995). The lower

region of the hind limb (tibia) was prepared for

aseptic surgery followed by routine orthopedic

operative draping and gowning procedures. A

prophylactic course of Cefotaxime sodium

(Cefotax®: EPICO, ARE) at dose of 4.5

mg/kg/bw was administered intravenously before

the operation and repeated every 8 hours for five

successive days post operation.

2.4.2. Surgical procedure (Fig. 1 A & B)

A 10-cm skin incision was made at the proximal

third of the medial surface of the right tibia. The

incision includes skin and periosteum to expose

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the bone (Fig. 1A) (MacNeill et al., 1999). Two

10 mm diameter holes were created at the

proximal third of the tibia. They were 1cm apart

from each other. Each defect extended through

only one cortex. The drilled holes were packed

using sterile gauze to control hemorrhage from the

medullary cavity. The previously prepared

WJSCs suspension was added to the first hole at

the time of the operation using micropipette while,

the second one was left empty as control negative

(Fig. 1B). The surgical wound was closed using

polyglactin 910 (Vicryl®).

2.5. Post- operative follow-up evaluations

2.5.1. Clinical evaluation

All operated animals were subjected to daily

clinical examination, including appetite, wound

drainage, local reaction, regional lymph node size

and the wound status.

2.5.2. Laboratory assessment of inflammation

Blood samples were collected directly before the

operation and at time points 1, 2, 3, 4 weeks post

operation to evaluate post- operative

inflammation. Inflammatory markers including

total leukocytic count (TLC), erythrocyte

sedimentation rate (ESR) and quantitative

measurement of C-reactive protein (CRP) were

measured. TLC and ESR were evaluated using the

classical Westergren method (Westergren,

1957), while, CRP was evaluated using

immunoturbidimetric method (Peltola, 1982).

2.5.3. Radiological evaluation

Medio-lateral views were taken on a standard

30×40 cm film at 50-52 kVp, 85 FFD and 10 mAs

by using X-ray apparatus (Semens 300). First

radiographs were taken immediately post-

operation and once every 2 weeks until the end of

the study (six months). Radiographs were

evaluated for radiographic density of the

implanted and the control holes.

2.5.4. Morphological studies

At the end of each observation period 1st, 3rd and

6th months. The dogs of each group were

euthanised using large dose of thiopental sodium.

The operated tibiae were harvested and examined

grossly. Each hole was inspected for complete or

partial filling in relation to the adjacent bone.

2.5.5. Histopathological Examination

Bone samples were collected from the operated

tibiae. Each sample contain the defect hole with

its surrounding healthy tissue was immediately

fixed in 10% formalin for one week. The samples

were decalcified using10% EDTA di-sodium

solution (P.93®: El Nasr pharmaceutical

chemical, Egypt) for one month (Shibata et al.,

2000). Decalcified samples were routinely

prepared and impeded in paraffin wax. A 3-5-

micron sections were mounted on glass slides,

deparaffinized, rehydrated and stained with

hematoxylin and eosin for histopathological

examination

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Fig. 1: Intraoperative photograph showing: A, the site of the incision at the proximal tibia. B the

drilled holes for implantation of WJSCs

RESULTS

Isolation and Immunophenotypic

characterization of WJSCs:

Nine from fifteen samples were successfully

isolated with viability 96-100%. It showed

adherence to plastic surface and fibroblastoid

morphology reaching 70%-80% confluence at day

15- 21 of culture (Fig. 2). Using flowcytometry,

the cells were positive for CD Oct3/4, Cd44 and

negative or low expression to CD34 (Fig. 3 &

Table 1).

Clinical assessment:

There is no evidence of infection or seromal

reaction in the operated animals as, assessed by

visual and palpable monitoring the degree of

swelling and temperature of the operated limbs,

regional lymph node size as well as, absence of

wound drainage.

Laboratory results of inflammatory markers

TLC, ESR and CRP were increased from basal

level before the operation to postoperative periods

1st and 2nd weeks and begin to decrease at the 3rd

week then returned to its normal levels by the end

of the 4th week (Table 2)

Radiographic results:

On examination of the radiographs taken for the

operated tibia directly after the operation and

throughout the observation period, it was noticed

that; both control hole and hole implanted with

WJSCs appeared radiolucent immediately post-

operation (Fig. 4A). By the end of the 2nd week

post operation, a slight radiopaque zone at the

hole’s margin implanted with WJSCs was noticed,

while the control one showed no detectable

changes (Fig. 4B). By the end of the fourth weak

post operation; the holes implanted with WJSCs

showed irregularity at its margins with slight

reduction in its width compared with previous

period. At this period, the control hole showed a

slight increase of radiopacity at its margin (Fig.

4C). At 12th weeks post operation, the holes

implanted with WJSCs showed marked reduction

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in its diameter compared to the control ones (Fig.

4D). At 24th week post operation the holes

implanted with WJSCs was difficult to be detected

radiographically, while the control one could be

easily detected with a marked radiolucent zone at

its center (Fig. 4E).

Histopathologic results

Microscopical examination of bone samples

collected from control group euthanized after one

month and three months’ post-operation showed

that, the entire bone holes were filled with

collagen bundles (Fig. 5A & B). After six months’

post-operation, control bone-samples

begins to form immature bone cells mixed with

collagen bundles (Fig. 5C). In the other hand,

bone holes treated with WJSCs showed that the

entire bone holes were filled with organized

fibrous connective tissue with newly formed

capillaries at one-month post-operation (Fig 5D).

At three months’ post-operation immature bone

cells mixed with collagen bundles were observed

(Fig. 5E). Well-developed mature bone matrix

mixed with remnant of immature bone matrix

were detected at 6th month post-operation (Fig,

5F).

Figure 2: Wharton jelly derived MSCs showed fibroblastoid adherent cells at 15 day (a) with 80%

confluence appearance at 21 day (b).

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Figure 3: Mesenchymal stem cell markers by flowcytometry showing positive expression for CD44

and oct 3/4 and negative for CD34

Table 1: Percentage expression of Wharton jelly MSCs markers by flowcytometry

The studied group

N= 9

Okt3/4 CD44 CD34

Range (%) 53.5-68.7 43.5-63.0 1.4-4.7

± SD 61.57±5.6 54.68± 6.32 3.4± 1.15

Median 59 57 3.7

X

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Table 2: Inflammatory markers of the studied groups basal level and postoperative at different time

points

ESR CRP Total leukocytic

count

The studied group

N= 9

10.0- 20.0

14.4 ± 3.39

2.0-5.0

3.88± 1.0

4.2-13.8

7.54±2.93

Base level

Range

X ±SD

40- 60

50.33± 4.8

24- 36

29.1± 4.8

14.5- 24.8

20.22± 3.25

1st week

Range

X ±SD

28- 45

34.22± 5.51

12- 20

14.55± 3.57

7.8- 11.4

9.84 ±1.37

2nd week

Range

X ±SD

10-20

16.33±3.2

6-15

11.0± 2.8

7.6- 11.1

9.73 ±1.13

3rd week

Range

X ±SD

3-15

9.0 ±4.0

3-8

4.88± 1.61

4.8- 9.8

7.53 ±1.59

4th week

Range

X ±SD

DISCUSSION

Several techniques of bone reconstruction have

been used to treat different bone affections,

especially that resulted in bone loss (Torroni,

2009; Kim et al., 2009; Nandi et al., 2010; Heo

et al., 2011; Emara et al., 2013a&b). The current

techniques are directed toward enhancement of

bone ability to regenerate itself. This was

achieved by using cells capable of bone formation

when delivered to a skeletal defect, thus avoiding

the need for harvesting bone (Pountos et al.,

2007; Jang et al., 2008; Jafarian et al., 2008

Chen et al., 2009; Udehiya et al., 2013).

Although the use of autogenic mesenchymal stem

cells provides high quality repair without the risk

of immune rejection (Udehiya et al., 2013), the

increased rate of donor site morbidity and the

prolonged time required for their expansion

before implantation are the most common

limitation of their wide use (Kim et al., 2007;

Watson et al., 2014).

In the present study, we have focused on

characterization of MSC populations originating

from the Wharton’s jelly matrix of human fetal

umbilical cord (UC) termed WJSC and evaluate

their ability to repair critical size gap defect in

canine model. As well as to proof that stem cells

has the ability to overcome host immune system

especially when they implanted xenogenically.

We isolate nine from fifteen samples, six samples

failed to be completed because of contamination.

The isolated cells have fibroblastoid morphology

and express the immunophenotypic markers

consistent with MSCs. This MSC source has

many advantages over other stem cell sources in

terms of: this source has abundant cell

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availability, relative ease of access for stem cell

isolation with minimal or no tissue morbidity, as

well as, the isolated cells has an immunogenic

profile favorable for cellular transplantation

(Puissant et al., 2005; Weiss et al., 2008).

In this study the used experimental animals

(dogs) may give us an idea about the nature of

the repair process in human because of

similarity of the biological repair process

between dogs and human (Burchardt, 1987).

Fig. 4: Showing sequential radiography of the operated tibia: A) at day zero. B) two weeks’ post-

operation. C)1month post-operation. D) 3 months post-operation. E) 6 months post-operation. The

hole number (1) is implanted with stem cells while hole number (2) left empty as control one.

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Fig. 5: Dog, tibial bone. A, B & C representing control group at 1, 3 & 6 months’ post operation,

respectively. A & B) showing collagen bundles filled the entire bone holes (asterisks). C) showing

that the defected area begins to form immature bone cells (arrow) mixed with collagen bundles

(asterisk). D, E & F representing WJSCs treated group at 1, 3 & 6 months’ post operation, respectively.

D) showing that the entire hole filled with organized fibrous connective tissue (asterisk) with newly

formed capillaries (arrows). CB, compact bone. E) showing that the defected area filled with

immature bone cells (thin arrow), collagen bundles (asterisk) and blood vessels (thick arrow). F)

showing well-formed mature bone (arrow) in between remnant of immature bone. H &E stain, X 10.

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In our study, we choose the end of the

observation period (six months) depending on the

radiographic evaluation results. By the end of the

observation period the WJSCs implanted hole

completely disappeared and couldn’t be detected

radiographically compared to the control one,

which indicated new bone formation regardless

the quality of the newly formed bone (Arinzeh et

al, 2003). The histological findings complimented

the radiological results and indicated new bone

formation in the hole implanted with WJSCs by

the end of the observation period as compared to

control holes. The increased osteogenesis may be

attributable to the conversion of stem cells into

osteoblasts then into osteocytes that responsible

for tissue formation and mineralization (Jang et

al., 2008; Jiang et al., 2010; Udehiya et al.,

2013), as well as, their ability to secrete several

growth factors which might have accelerated

healing (Chen et al., 2009). Concerning the

quality of the newly formed bone, the hole

implanted with WJSCs showed mature bone

formation interspersed with small amounts of

immature bone remnants. According to the

authors opinion, the quality of the newly formed

bone can be improved by prolongation of the

observation period or by using a scaffolding

material to enhance the development and

migration of the stem cells, but this wasn’t

proofed here and require further investigation.

The measured inflammatory markers here showed

marked increase directly after the operation and

during the post-operative period till the end of the

3rd week and returned to its normal level by the

end of the 4th week. This may be attributed to

traumatic injury during the operation. According

to our results there is no any immune response

provoked against WJSCs when they were

implanted xenogenically in dogs, this result found

in accordance with previous reports published by

(Guo et al., 2009; Udehiya et al., 2013) whom

stated that mesenchymal stem cells have little or

no immunogenicity when they used in bone

healing. It seems that mesenchymal stem cells

overcome immune rejection by three broad

mechanisms including: (1) absence of major

histocompatibility complex II (MHC II) on their

cell surface, which responsible for the antigenicity

of the cells, (2) direct and indirect prevention of T

cell responses and (3) induction of suppressive

local microenvironment (Di Nicola et al., 2002;

Ryan et al., 2005; Uccelli et al., 2006; Nauta and

Fibbe 2007; Planka et al., 2008; Guo et

al.,2009; Jung et al., 2009). In conclusion; this

study supported the hypothesis that mesenchymal

stem cells have no any detectable immune

reaction and can be used xenogenically in animals

to enhance and accelerate bone healing.

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