The effect of deferoxamine injection on composite graft survival in rabbits

Abstract

Background
Composite graft as a reconstructive therapy option has limitations in size so that it is easily necrotic. Deferoxamine administration has been associated with increased neo-vascularity in wounds. We aimed to compare the administration of deferoxamine and Platelet-Rich Plasma (PRP) injection in a composite graft in rabbits.

Methods
Thirty New Zealand rabbits were divided into three groups; the control group, the deferoxamine group, and the PRP group. The composite graft with a diameter of 2 cm was taken and replanted after rotating it 180°. The mean graft viability and the mean number of capillaries were evaluated on day 7 (POD 7) by macroscopic and histological evaluation using Hematoxylin-Eosin staining.

Results
While the mean number of capillaries was not significantly different in control, deferoxamine, and PRP groups (p = 0.21), the mean survival rate in the control, deferoxamine, and PRP groups reached a significant level with p-value of 0.006 (66.6% vs. 63.8% vs. 99.6%, respectively).

Conclusions
Deferoxamine group had the highest number of capillaries, but had the lowest survival rate. In the PRP group, it had the lowest number of capillaries, but had the highest survival rate.

Full text

Annals of Medicine and Surgery 60 (2020) 542–545
Available online 21 November 2020
2049-0801/© 2020 The Authors. Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Experimental Research
The effect of deferoxamine injection on composite graft survival in rabbits
Rianto Noviady Ramli
a
,
*
, Agus Santoso Budi
b
, Sitti Rizaliyana
b
, Aditya Rifqi Fauzi
a
a
Plastic Reconstructive, and Aesthetic Surgery Division, Department of Surgery, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito
Hospital, Yogyakarta, 55281, Indonesia
b
Department of Plastic Reconstructive, and Aesthetic Surgery, Faculty of Medicine, Universitas Airlangga/Dr. Soetomo Hospital, Surabaya, Indonesia
ARTICLE INFO
Keywords:
Composite graft
Survival rate
Deferoxamine
PRP
ABSTRACT
Background: Composite graft as a reconstructive therapy option has limitations in size so that it is easily necrotic.
Deferoxamine administration has been associated with increased neo-vascularity in wounds. We aimed to
compare the administration of deferoxamine and Platelet-Rich Plasma (PRP) injection in a composite graft in
rabbits.
Methods: Thirty New Zealand rabbits were divided into three groups; the control group, the deferoxamine group,
and the PRP group. The composite graft with a diameter of 2 cm was taken and replanted after rotating it 180◦.
The mean graft viability and the mean number of capillaries were evaluated on day 7 (POD 7) by macroscopic
and histological evaluation using Hematoxylin-Eosin staining.
Results: While the mean number of capillaries was not signicantly different in control, deferoxamine, and PRP
groups (p =0.21), the mean survival rate in the control, deferoxamine, and PRP groups reached a signicant
level with p-value of 0.006 (66.6% vs. 63.8% vs. 99.6%, respectively).
Conclusions: Deferoxamine group had the highest number of capillaries, but had the lowest survival rate. In the
PRP group, it had the lowest number of capillaries, but had the highest survival rate.
1. Background
Reconstruction of physical defects or deformities due to several
conditions, either congenital or acquired back into normal form and
function, is the main goal of plastic and reconstructive surgery. Com-
posite grafts yield better results than skin grafts in discrepancies in color
and texture at the donor site, causing less scar contracture, because more
tissue structure is provided by transferring 2 or more different tissue
types. However, the composite graft has limited graft viability when the
recipient defect is more than 1.5 cm in diameter [1].
With the limitation of re-vasculature, the failure rate of composite
graft is quite high [2–4]. Angiogenesis which plays a role in graft
re-vascularization can be increased by direct injection of growth hor-
mones such as broblast growth factor (FGF), platelet derived growth
factor (PDGF) and vascular endothelial growth factor (VEGF). The
important role of VEGF in angiogenesis is as a specic mitogen factor for
endothelial cells, which stimulates the formation of new blood vessels
and increases their permeability. In PRP, there are many of the growth
factors mentioned above which are proven to increase the viability of
the composite graft [5].
However, the availability of PRP in local health services is an
obstacle. Therefore, the use of alternative drugs needs to be considered.
Deferoxamine as an iron chelating agent has been shown to have a
wound healing effect [6,7]. We aimed to compare the administration of
deferoxamine and Platelet-Rich Plasma (PRP) injection in a composite
graft in rabbits.
2. Material and methods
2.1. Animal models
Thirty male New Zealand rabbits, aged 9–12 months, weighing
2500–3000 g were isolated under standard conditions for 7 days. Rab-
bits that were sick, had an infection during the procedures, or died
during the procedures were excluded from this study. The work has been
reported in line with the ARRIVE guideline [8].
* Corresponding author. Plastic Reconstructive, and Aesthetic Surgery Division, Department of Surgery Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada Dr. Sardjito Hospital, Yogyakarta, 55281, Indonesia.
E-mail addresses: antobedah28@gmail.com (R.N. Ramli), agus_sbeyk@yahoo.com (A.S. Budi), srizaliyana@gmail.com (S. Rizaliyana), aditya.rifqi.f@mail.ugm.ac.
id (A.R. Fauzi).
Contents lists available at ScienceDirect
Annals of Medicine and Surgery
journal homepage: www.elsevier.com/locate/amsu
https://doi.org/10.1016/j.amsu.2020.11.029
Received 27 October 2020; Received in revised form 5 November 2020; Accepted 7 November 2020

Annals of Medicine and Surgery 60 (2020) 542–545
543
2.2. Experimental procedures
All experiments were performed in the Faculty of Veterinary Medi-
cine of Universitas Airlangga. These experimental procedures were
performed with the prior approval of the Medical and Health Research
Ethics Committee of the Faculty of Medicine, Universitas Airlangga (2.
KE.131.07.2018). Each rabbit was injured in the ear with a diameter of
2 cm, full thickness through and through. Then the composite graft on
the rabbit ears consisting of skin tissue, sub cutis, and rabbit auricula
cartilage in the form of a circle 1 cm in diameter was excised from the
rabbit auricula, rotated 180◦, and sewn back (Fig. 1).
In group I, no subcutaneous injection was given. Group II was given
subcutaneous 100 mg/kg deferoxamine injection (Novartis Pharma
Stein AG Stein, Switzerland), in all four quadrants outside the composite
graft using a 1 cc injection syringe one day before the composite graft,
post graft treatment procedure, and within the rst 3 days. Group III was
given PRP injection of 0.5 ml subcutaneous, post graft, and within the
rst 3 days. The viability of the graft was checked every day and the 7th
day was assessed with Visitrak (Kyros International Inc., USA). On the
7th day, the skin was harvested for examination of vascular density.
Composite graft was assessed for its viability based on macroscopic
observations, the non-viable tissue was described by the presence of
necrotic, darker color accompanied by dry tissue, such as tanning in the
red marker areas in Fig. 2, while the viable graft ngers were not visible
as green areas.
Fig. 1. Composite graft treatment scheme.
Fig. 2. Composite graft treatment viability.
R.N. Ramli et al.

Annals of Medicine and Surgery 60 (2020) 542–545
544
2.3. Statistical analysis
Kruskal-Wallis and Mann-Whitney test were used to evaluate the
graft survival between groups. Independent t-test was used to evaluate
the capillary count between groups. For statistical analyses, a p-value of
less than 0.05 was considered to be signicant and 95% condence in-
terval was used in this study.
3. Results
The graft survival of rabbits between control vs. deferoxamine vs.
PRP reached a signicant level (66.6% vs. 63.8% vs. 99.6%; p =0.006).
Further analysis showed that control vs. PRP, and deferoxamine vs. PRP
differ signicantly with p-value of 0.003 and 0.004, respectively
(Table 1).
However, in terms of capillary counts, no signicant difference was
found between control vs. deferoxamine (204.7 ±44.4 vs. 212.8 ±98.2,
p =0.82), and deferoxamine vs. PRP (212.8 ±98.2 vs. 160.8 ±39.9, p =
0.147). But, in contrast, control vs. PRP showed a signicant difference
(204.7 ±44.4 vs. 160.8 ±39.9, p =0.042) (Table 2).
4. Discussion
Deferoxamine group has the lowest graft viability rate with only
63.8%, this is in contrary to the results obtained by Wang’s [9] study,
namely the injection of deferoxamine in mouse back skin aps, which
was started 1 day before and continued for 3 days after ap elevation is
90.1% at a dose of 100 mg/kg.
The viability of a composite graft depends on several factors, such as
skin grafts. The viability of the composite graft comes from serum in-
hibition, re-anastomosis of blood vessels, and neovascularization [10].
However, unlike skin grafts, composite grafts have a layer of cartilage,
which can act as a mechanical barrier that limits the vascularization of
the wound bed. Thus, it is conceivable that revascularization via the
dermis connection to the dermis at the wound edge is more important
for the survival of the composite graft, and in turn limits the size of the
composite graft [11]. Because it is like a skin graft, the viability of the
composite graft is inuenced by good recipient vascularity, accurate
contact between the graft and recipient, and immobilization [12].
Several conditions that can reduce the viability of the composite
graft, such as the pressure on the composite graft can damage the
recanalization and neovascularization process, this is because the pres-
sure makes the ischemic tissue, ischemic tissue releases inammatory
mediators that cause edema, moreover will cause vascular thrombosis
[10]. On the basis of this, the low viability of the composite graft in the
deferoxamine group can occur due to the large number of substances
injected into the ears of rabbits, it has a body weight of 2 kg, based on
the dose of 100 mg/kg each ear and the deferoxamine content of 100
mg/cc, so each ear gets 2 cc, or each quadrant received 0.5 cc of
deferoxamine. During this treatment, 0.5 cc which is injected into the
subcutaneous can create bulging and edema in the recipient subcu-
taneous layer, which will interfere with the vascular inosculation pro-
cess from the recipient wound edge to the wound edge of the composite
graft, so that the process has an effect on the revascularization process
that is not working well and the end result will affect the viability of the
composite graft. This mechanism was repeated four times according to
the schedule of administration in the deferoxamine treatment group up
to day 7.
The high viability results were in the PRP group, which had a mean
graft viability of 99.6%. This is in line with Choi’s [5] study conducted
on composite rabbit ear grafts, namely that PRP administration to the
recipient area 3 days before grafting had a graft survival rate of 97%,
compared to administration immediately after graft (69.2%), 3 days
after graft (55.7%) and control (40.7%).
Platelet-rich plasma (PRP) contains various growth factors such as
platelet derived growth factor (PDGF), transforming growth factor - β
(TGF-β), vascular endothelial growth factor (VEGF), endothelial growth
factor (EGF), insulin-like growth factor (IGF1), endothelial cell growth
factor (ECGF) [13]. The growth factors released by PRP can increase
epithelialization, the amount of collagen, wound strength, epidermal
regeneration, stimulate angiogenesis, accelerate homeostasis, therefore
the use of PRP can increase skin ap viability [14–16]. The use of PRP
can increase tissue regeneration and reduce the risk of infection, pain,
and blood loss. In addition, PRP may suppress the release of cytokines,
suppress inammation, and will increase tissue regeneration. Angio-
genesis is known to take 3–5 days [10].
Despite having a low mean graft viability in the deferoxamine
treatment group, the histological ndings in the form of capillary count
were the highest of the other groups, although statistically there was no
signicant difference. Thangarajah [17] states that deferoxamine makes
HIF-1
α
stable in wounds, which in turn stimulates the expression of
VEGF which ultimately increases neo-vascularization. Vascular endo-
thelial growth factor (VEGF) is the permeability factor of blood vessels
released from the wound epithelium and extracellular matrix by pro-
teases from endothelial cells, stimulating endothelial cell proliferation
and increasing vascular permeability. This affects plasma protein
extravasation and creates a temporary support structure through which
activated endothelial cells, leucocytes and epithelial cells can further
migrate [18]. Therefore, the number of capillaries was more in the
deferoxamine group.
The results differed in the PRP group, the number of capillaries in the
PRP group was the least, this could be due to the wound healing process
running very well. Li [19] found a signicant increase in PDGF
expression after 8 h, and lasted from 3 to 7 days. The release of PDGF
into the skin can have a chemotactic effect on monocytes, neutrophils,
broblasts, mesenchymal stem cells. Platelet derived growth factor
(PDGF) is also a strong mitogen for broblasts and smooth muscle cells
and is involved in the wound healing phase (i.e., angiogenesis, brous
tissue formation, and re-epithelialization) [15]. The histologic ndings
of the PRP group appear that granulation tissue is reduced and vascular
Table 1
Graft survival analysis.
Treatment group N Mean rank p
Graft survival Control 8 10.75 0.006*
Deferoxamine 10 11.25
PRP 10 20.75
Graft survival Control 8 9.38 0.928
Deferoxamine 10 9.60
Graft survival Control 8 5.88 0.003**
PRP 10 12.4
Graft survival Deferoxamine 10 7.15 0.004**
PRP 10 13.85
PRP: Platelet-rich plasma, *p <0.05 is considered signicant by Kruskal-Wallis,
**signicant by Mann-Whitney.
Table 2
Capillary count analysis.
Treatment group N Mean ±S.D p
Graft survival Control 8 204.7 ±44.4 0.209
Deferoxamine 10 212.8 ±98.2
PRP 10 160.8 ±39.9
Graft survival Control 8 204.7 ±44.4 0.82
Deferoxamine 10 212.8 ±98.2
Graft survival Control 8 204.7 ±44.4 0.042*
PRP 10 160.8 ±39.9
Graft survival Deferoxamine 10 212.8 ±98.2 0.147
PRP 10 160.8 ±39.9
PRP: Platelet-rich plasma, S.D: standard deviation, *p <0.05 is considered
signicant using independent t-test.
R.N. Ramli et al.

Annals of Medicine and Surgery 60 (2020) 542–545
545
tissue is also reduced, which is a sign that the tissue is healing.
Our study is not without limitation. First, we did not measure level of
the growth factor to see the effect of injection of deferoxamine and PRP
on the composite graft. Second, we did not perform histological exam-
ination on the 3rd day after the graft as a comparison to prove that the
PRP administration also increase the number of capillaries before
experiencing a decrease in the number on the 7th day. Therefore, further
study with larger sample is necessary to conrm our ndings.
5. Conclusions
Administration of deferoxamine three days before grafting can
signicantly increase the viability of composite graft. Our study implies
that the usage of deferoxamine might have benecial effect on com-
posite graft viability.
Provenance and peer review
Not commissioned, externally peer-reviewed.
Ethical approval
Not applicable.
Sources of funding
The authors declare that this study had no funding source.
Author contribution
Rianto Noviady Ramli conceived the study and approved the nal
draft. Aditya Rifqi Fauzi drafted the manuscript. Agus Santoso Budi and
Sitti Rizaliyana critically revised the manuscript for important intel-
lectual content. All authors read and approved the nal draft. All authors
facilitated all project-related tasks.
Trial registry number
Not applicable.
Guarantor
Rianto Noviady Ramli.
Consent
Not applicable.
Declaration of competing interest
No potential conict of interest relevant to this article was reported.
Acknowledgements
We thank all staff members for assistance during the study.
Abbreviations
POD Postoperative day
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.amsu.2020.11.029.
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