Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant

Acta Scientiae Veterinariae, 2016. 44: 1355.

RESEARCH ARTICLE

Pub. 1355

Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant

ISSN 1679-9216
1
Received: 2 October 2015 Accepted: 25 April 2016 Published: 14 May 2016
1
Programa de Pós-graduação em Ciências Veterinárias (PPGCV), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. 2
Laboratório
de Embriologia e Diferenciação Celular, Hospital de Clínicas de Porto Alegre (HCPA)-UFRGS, Porto Alegre. 3
Unidade de Experimentação Animal,
Centro de Pesquisa Experimental, HCPA-UFRGS. 4
Departamento de Patologia Clínica Veterinária, FaVet-UFRGS. 5
Departamento de Medicina Animal,
Faculdade de Veterinária, FaVet-UFRGS. CORRESPONDENCE: E.O. Cirne-Lima [ecirnelima@gmail.com – Fax: +55 (51) 3308-7305]. Faculdade de
Veterinária – UFRGS. Av. Bento Gonçalves n.9090, Bairro Agronomia. CEP 91540-000 Porto Alegre, RS, Brazil.
Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant
Plasma Rico em Plaquetas em as frações líquida e em gel combo adjuvants da cicatrização cutânea
Tulane Nerissa Alves Garcez1,2, Helena Flores Mello2
, Priscilla Domingues Mörschbacher1
,
Paula Barros Terraciano2
, Vivian Pignone1
, Marta Justina Cioato3
,
Elizabeth Obino Cirne-Lima2,4 & Emerson Antonio Contesini5

ABSTRACT

Background:

In recent decades, many types of research have been conducted on processes involved in tissue repairing, mainly in
the development of resources and technology designed to improve the wound healing progress. Platelet-rich plasma (PRP)
derived from autologous blood is defined as a plasma volume with platelet concentration higher than physiological level. It
is an autogenous and low-cost source of growth factors, which are essential for tissue regeneration due to their angiogenic,
mitogenic, and chemotactic properties. The aim of this study was to evaluate two forms of PRP- liquid, and gel – regarding their
capacity to influence the quality and repair time of standardized skin injuries.

Materials, Methods & Results:

New Zealand healthy rabbits were distributed in three groups (n = 6): control group (CG),
liquid platelet-rich plasma group (LIQPRP), and gel platelet-rich plasma group (GELPRP). Acute skin lesions were inducted
in two areas approximately 2 cm close to scapular edge and depth including epidermis, dermis, and hypodermis to the external
muscular fascia. Animals received treatment according to each group. Injuries were measured with digital pachymeter in
two directions: longer length (l) and longer width (w), every two days. Areas and healing rates were calculated. Microscopic
analysis samples were collected on days seven and 14 and evaluated through hematoxylin and eosin staining (HE) for global
tissue examination, and through Masson’s trichrome (MT) to collagen fibers present within the interstice. These analyzes considered:
angiogenesis, inflammation infiltrated and collagen fibers quantity. Immunohistochemistry with anti-Ki-67 antibody
was utilized for proliferative profile assessment. Kruskal-Wallis’ non-parametric tests of independent samples were performed
for comparison of values obtained through platelet count, referring to the evaluation of platelet increase on treatments. Scar
contraction rate (CR) was evaluated through the Shapiro-Wilks normality test and then submitted to mixed models test. Results
obtained by histopathological and immunohistochemistry were also evaluated by Shapiro-Wilk’s normality test (for all tests
a 5% level of significance was considered). Platelet concentration achieved with liquid PRP was 8.64 and gel PRP reached
5.62 times higher than physiological values. Platelet increase mean for both groups was 7.95. No statistical significance was
observed between groups. No side-effects or adverse reactions related to PRP usage were observed while the study was conducted.

Discussion:

In the present study, there was a need to raise platelet-poor plasma volume in order to obtain autogenous thrombin
required for gel PRP. After this modification, a stable and reasonable platelet concentration gel was produced. However, this
form of PRP application requires more time for sample preparation, increasing the production cost. Furthermore, injection of
liquid PRP directly in the wound site activates platelets by generated substances due to needle perforation, and mainly due to
tissue trauma generated at the lesion site. Relating to the therapies administered, gel PRP was considered more manageable,
since 3D structure could easily adapt to wound site after simply deposition of it. Liquid PRP was administered with needle
and syringe, which required the surgeon to be more careful and perform a slow injection in order to avoid any spill and loss
of material. Furthermore, the histopathological analysis did not point any clot traces formed by gel PRP dehydration, although it
is not possible to ensure that the clot was eliminated, reabsorbed, or even removed by the animal. By this protocol, a stable
and reasonable platelet concentration gel was produced. Further studies are encouraged as well as employment of alternative
diagnostic tools, in order to better understand found results.

Keywords:

skin, regenerative therapy, cell therapy, growth factors, platelets.

Descritores:

pele, terapia regenerativa, terapia celular, fatores de crescimento, plaquetas.
2
T.N.A. Garcez, H.F. Mello, P.D. Mörschbacher, et al. 2016. Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant.
Acta Scientiae Veterinariae. 44: 1355.

INTRODUCTION

Platelets (PL) are blood cells that play an
important role in homeostasis and are also one of the
main sources of growth factors (GF). Several studies
have shown that application of autogenous concentrates
derived from platelet or platelet-rich plasma (PRP)
within a lesion area can accelerate tissue repair. This
the study aims to evaluate PRP usage in two forms: liquid
and gel, in relation to their ability to influence the quality
and repair time of standard skin injuries in New
Zealand rabbits.

MATERIALS AND METHODS

This research utilized 18 New Zealand healthy
female rabbits, aged between four and five months and
body mass between two and three kilograms, from
a breeder registered to Unidade de Experimentação
Animal from Hospital de Clínicas de Porto Alegre
(UEA-HCPA).
PRP Centrifugation Protocol
Subsequently to pilot studies [6] a PRP
preparation protocol was selected for the application.
Venipuncture of jugular vein was utilized for blood
sample collection, due to the high volume needed. Thus,
punction area was prepared with animals anesthetized,
and 15 mL of blood was collected with a 25×7 needle
attached to 10 mL syringe filled with 1 mL sodium
citrate anticoagulant. The the blood sample was stored in
four flasks with 0.5 mL sodium citrate each and was
immediately sent to Laboratório de Embriologia e
Diferenciação Celular/HCPA for processing. One mL
from whole blood sample was saved for platelet and
other cellular counts. All procedure was conducted in
laminar flow hood in order to keep an aseptic sample
since the product would be administered to animalThe protocol
col utilized for PRP obtaining proposes
two centrifugations (Eppendorf®)
1
, the first one at 250 g
for 10 min. After that, the sample had three distinct layers:
the lower layer, containing red blood cells, the intermediate
layer, formed by a “cloudy zone”, that included leukocytes
and larger platelets, and the upper layer comprising
by the plasma. Cloudy zone and plasma were collected,
and the remaining volume was discarded. Collected
fraction was submitted to a second centrifugation at
1000 g for 10 min. By the end of this procedure, a sample
showed a pellet and the supernatant that was composed
by plasma. Pellet was collected along with 150 µL from
plasma, which would both form PRP product after homogenization.
Product was transferred to a microfuge
tube and delivered to a surgeon for an immediate transplant.
Gel PRP was obtained following all steps
above until the end of second centrifugation. Then,
pellet was collected along with 2 mL of supernatant. A
0.5 mL aliquot was destined to platelet count and 1,5
mL aliquot was divided in two portions and transferred
to 24-well plates, used as circular templates, and 150
µL of calcium gluconate was added for coagulation and
gel formation. Products were sent to the surgery center for
immediate transplantation.
Platelet count was conducted at blood collection
time and by the end of each concentration protocol
in all samples, by automatic method. Platelet quantity
reached in each sample and basal values were both
analyzed.
Surgical Procedure
Animals under general anesthesia had the dorsal
skin region delimitated in two areas approximately
2 cm close to the scapular edge, about 6 cm apart from
each other. Delimitated areas had the skin removed
and injury depth including epidermis, dermis, and
hypodermis to the external muscular fascia.
Therapeutic procedures
Animals were randomly divided in three
groups (n = 6): control group (CG), liquid platelet rich
plasma group (LIQPRP), and gel platelet rich plasma
group (GELPRP). Immediately after injury assessment,
animals received the treatment according to the group they
composed (Figure 1).
Sample obtaining
After 7 or 14 days period, animals were
submitted to a new surgery for total lesion area removal,
including 2 mm beyond the boundary of scar and
neighboring healthy tissue. Scar from the left side was
removed at day 7 of post-surgery, and right side scar
was collected at day 14 of post-surgery. Fragments
were fixed in 10% formaldehyde, embedded in paraffin
and processed.
Morphometric evaluation
Injuries were measured with a digital pachymeter
in two directions: longer length (l) and longer width
(w), on days 0, 2, 4, 6, 7, 8, 10, 12, and 14. These data
allowed the calculation of lesion area in each evaluation.
Wound contraction area (C) was determined subtracting
3
T.N.A. Garcez, H.F. Mello, P.D. Mörschbacher, et al. 2016. Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant.
Acta Scientiae Veterinariae. 44: 1355.
initial area (A1
) measured at day 0 (injury induction day),
from lesion measurements obtained in the following
evaluation days (x = 2, 4, 6, 7, 8, 10, 12, and 14), so
that C=AX–A1
. Scar contraction rate (CR) was assessed
based on previous results, where CR= C x 100/A1
.
Microscopic evaluations
Histological sections were stained by Hematoxylin
and Eosin (HE) technique, for global evaluation
of tissue slices, and by Masson’s Trichrome (MT), to
examine the presence of collagen fibers.
In order to compare the different treatments
we analyzed histological sections as follows: angiogenesis,
shown by granulation tissue, based on newly
formed vessels and fibroblasts; inflammatory infiltrate,
by presence and quantity of mononuclear and polymorphonuclear
cells; and collagen fibers quantity.
Proliferative profile evaluation was performed by
immunohistochemistry analysis with an anti-Ki-67 antibody.
Analyzes were evaluated by categorical variable:
strong, moderate, and weak.
Statistical Analysis
Kruskal-Wallis’ non-parametric tests of independent
samples were utilized for comparison of values
obtained through platelet count, referring to the evaluation
of platelet increase on liquid and gel PRP treatments.
Scar contraction rate (CR) was evaluated through
Shapiro-Wilk’s normality test, and then submitted to
mixed models test. Obtained results by histopathology
and immunohistochemistry analysis were compared by
the same test. All tests considered a significance level
of 5% (P < 0.05).

RESULTS

Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant
Platelet count
All animals treated with PRP, regardless of
the form (liquid or gel), had a basal platelet count
determined for further analysis of platelet increment
reached through concentration protocol. Platelet
concentration achieved with liquid PRP was 8.64
times higher than physiological values (mean of
basal platelet count from all samples), and gel PRP
reached 5.62 times more platelet concentration than
physiological values. Platelet increase mean for both
groups was 7.95. No statistical significance was observed
between groups.
Morphometric evaluation
Measurements at days 0, 2, 4, 6, 7, 8, 10, 12,
and 14 allowed calculation of lesion area (larger length
versus larger width) and wound contraction area (C),
subtracting initial area (A1
) determined on day 0 (injury
induction day), from lesion area obtained on following
days of evaluation (x = 2, 4, 6, 7, 8, 10, 12, and 14), so
that C=AX – A1
. Scar contraction rate (CR) was evaluated
based on previous results, where CR= C x 100/
A1
(Figures 2 & 3).
Histopathological parameters (inflammatory
a reaction, collagen deposition, and angiogenesis) did
not show the difference in group means at days 7 and 14.
Immunohistochemistry parameters referring to epithelial
and fibroblast proliferation mean percentages revealed
the significant difference between LIQPRP group and CG
at day 14 (Figure 4).
Figure 1. Therapeutic procedures executed after skin injury induction in rabbits. (A) Liquid platelet rich plasma injection in 1 mL
final volume. (B) Gel platelet rich plasma administered on the wound site.
4
T.N.A. Garcez, H.F. Mello, P.D. Mörschbacher, et al. 2016. Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant.
Acta Scientiae Veterinariae. 44: 1355.
Figure 2. Average contraction rate (%) increase from each group “versus”
observation days. Difference between treatments can be observed until day
6, whereas by the end of evaluation there are no more differences.
Figure 3. Clinical evaluations. Lesions view on clinical evaluations at
days 7 and 14 post-surgery. At 14th day all groups exhibited similar rates
in injury healing.
Figure 4. Immunohistochemistry parameters (epithelial and fibroblastic proliferation). There is significant
difference between means of LIQPRP group and CG at evaluation day 14.

DISCUSSION

The main concern about the employment of a
product derived from the animal itself was due to the possibility
of immunological reaction associated to platelet
membrane antigens, which could lead to growth factors
secretion inability, and therefore in a therapeutic effect
failure. These circumstances were also addressed by
other studies [8]. Concerning the possibility of clinical
application, utilization of an autologous product can
avoid infectious diseases transmission, an important
point to be considered not only for animals but also
for human treatments [24].
PRP method utilized in this study was able to
achieve platelet concentration higher than 1.000.000
µL-1, increasing, on average, 7.95 times basal values.
This concentration is higher than the previous described
protocols [1,12,15,26], which could not reach three
times platelet concentration with patient’s blood.
Besides, we found higher concentration than those
obtained by automatic methods, that raised levels one
to four times physiological levels [11].
In the present study, there was a need to raise
platelet poor plasma volume (2 mL collected fraction)
in order to obtain autogenous thrombin required for gel
5
T.N.A. Garcez, H.F. Mello, P.D. Mörschbacher, et al. 2016. Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant.
Acta Scientiae Veterinariae. 44: 1355.
PRP. After this modification, a stable and reasonable
platelet concentration gel was produced. However, this
the form of PRP application requires more time for sample
preparation, since it needs to become a gel, therefore
increasing the production cost. Furthermore, injection
of liquid PRP directly in the wound site activates platelets
by generated substances due to needle perforation,
and mainly due to tissue trauma generated at the lesion
site [25].
Relating to the therapies administered, gel PRP
was considered more manageable, since a 3D structure
could easily adapt to wound site after simply deposition
of it. Liquid PRP was administered with the needle (0.7 X
25mm) and syringe (3 mL), which required the surgeon
to be more careful and perform a slow injection in order
to avoid any spill and loss of material.
The study did not show any side effects or adverse
reactions that could be attributed to PRP usage.
PRP gel usage demonstrated not to elongate inflammatory
phase and also not to produce any deleterious
changes, such as necrotic tissue and foreign body
reaction stimulus – these were previous concerns since
gel PRP was a 3D biomaterial transplanted to wound
site. Furthermore, the histopathological analysis did not
point any clot traces formed by gel PRP dehydration,
although it is not possible to ensure that the clot was
eliminated, reabsorbed, or even removed by the animal.
Literature does not indicate any conclusive information
regarding what happens to gel PRP fraction after its
effect within the tissue.
Despite wound contraction rate had shown
some improvement signals – i.e., elapsed time for healing
– for GELPRP group on initial days of evaluation
(2, 4, and 6), by the end of the experiment, there was no
difference between mean rates within treated groups.
At day 14 – last day of an experiment – all groups exhibited
similar rates in injury healing. Indeed, regardless of
several authors relates that they have found significant
improvements in tissue healing and bone repairing using
PRP, others did not notice the same benefits [15].
Different studies also could not demonstrate gel PRP
efficiency in the treatment of injuries in experimental
models [8,14,16]. Variations in some key features of
PRP including platelet concentration, and coagulation
activator, can alter PRP quality [2]. Platelet inability
to secret growth factors, or even early release of them
can be another reasonable explanation [4,11]. Morphological
analysis of platelets on a blood smear is highly
recommended as a tool to evaluate product quality,
because platelets have different morphology when
activated, and display cytoplasmic elongations [15],
however, it can cause a great delay in the product to deliver.
Moreover, a material could have been submitted to flow
cytometry [3]. Nevertheless, flow cytometry requires
specialists, limiting employment of this technique.
Inability of autogenous thrombin to activate platelets
is also a possibility that could explain PRP therapeutic
failure. To address this hypothesis, a study [19] suggested
that when autogenous thrombin is being utilized,
its concentration should be validated by the amidolytic assay.
This approach depends on thrombin quantification,
therefore specific equipment and materials are needed,
resulting in higher product cost and the addition of steps
for its achievement [20].
There is a hypothesis regarding growth factors
overload causing a dose dependent inhibitory effect. This
can be questioned since LIQPRP group has reached
unsatisfying results for contraction rate comparing to
GELPRP group. LIQPRP group reached a mean of
8.64, while GELPRP mean value was 5.62 times basal
values. Some studies analyzed PRP effectiveness in
increasing tissue repair, relating their results with several
administered platelet concentrations. Results have
shown that PRP effectiveness may follow concentration
dependence pattern, that is, really high concentrations
might cause prejudicial effect [21], however, there is
still no consensus about what increment levels would
be ideal and from what value there is no more benefit,
and more importantly, when the product starts being
prejudicial for the healing process.
CONCLUSIONS
Based on results obtained in this study, PRP
local administration did not accelerate the healing
process upon morphometric evaluation 14 days after
surgery, with the use of liquid PRP demonstrating lower
epithelialization levels by histopathological evaluation
compared to the Control group 14 days after surgery.
New studies exploring different diagnostic
tools are highly recommended in order to better understand
the results and provide improvements in current
treatments.
MANUFACTURER
1
Eppendorf® do Brasil Ltda., São Paulo, SP, Brazil.
6
T.N.A. Garcez, H.F. Mello, P.D. Mörschbacher, et al. 2016. Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant.
Acta Scientiae Veterinariae. 44: 1355.
Acknowledgements. This work was supported by Fundo de
Incentivo à Pesquisa (FIPE), Hospital de Clínicas de Porto
Alegre and Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq).
Ethical approval. All procedures were performed in accordance
with the 11.794 (08/10/2008) Experimental Animal Management
Brazilian Law. The study was approved by the Ethical
Commit of Hospital de Clínicas de Porto Alegre (CEUA –
HCPA, 11-0359).
Declaration of interest. The authors report no conflicts of
interest. The authors alone are responsible for the content and
writing of the paper.
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