Experimental Eye Research 203 (2021) 108402

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Experimental Eye Research

journal homepage: www.elsevier.com/locate/yexer

Development and optimization of a personalized fibrin membrane derived

from the plasma rich in growth factors technology
Eduardo Anitua a, b, *, María de la Fuente a, b, Francisco Muruzabal a, b, Jesús Merayo-Lloves c
a
Biotechnology Institute (BTI), Vitoria, Spain
b
University Institute for Regenerative Medicine and Oral Implantology - UIRMI (UPV/EHU-Fundación Eduardo Anitua), Vitoria, Spain
c
Instituto Oftalmológico Fernández-Vega. Fundación de Investigación Oftalmológica. Universidad de Oviedo, Oviedo, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Purpose: To develop and characterize a new type of plasma rich in growth factors (PRGF) membrane for patients
PRGF in which immune system is involved in the disease etiology.
Platelet-rich plasma Methods: Blood from three healthy donors was collected to obtain the different fibrin membranes by PRGF
PRP
technology. PRGF obtained volumes were activated and divided into two groups: PRGF membrane (mPRGF)
Fibrin membrane
Wound healing
obtained after incubation at 37 ◦ C for 30 min (control); and is-mPRGF: mPRGF obtained after incubation for 30
Autoimmune diseases min at 56 ◦ C. The concentration of several growth factors, proteins, immunoglobulin E and the complement
Ocular inflammatory diseases activity was determined in the different mPRGF. The proliferative potential of heat-inactivated mPRGF were
Heat-inactivation assayed on keratocytes (HK) and conjunctival fibroblasts (HConF). In addition, morphological and physical
features of the inactivated mPRGF were evaluated in contrast to the control mPRGF.
Results: Heat-inactivation of the mPRGF preserves the content of most of the growth factors involved in the ocular
wound healing while reducing drastically the content of IgE and the complement activity. The heat-inactivated
mPRGF conserve the morphological and physical characteristics of the fibrin meshwork in comparison with the
control mPRGF. Furthermore, no significant differences were found in the biological activity of the control
mPRGF regarding the heat-inactivated mPRGF (is-mPRGF) in any of both ocular cell types evaluated.
Conclusions: The heat-inactivation of the PRGF membranes (is-mPRGF) reduces drastically the content of IgE and
complement activity while preserving the content of most of the proteins and morphogens involved in ocular
wound healing. Furthermore, the morphological and physical features of the immunosafe mPRGF were also
preserved after heat-inactivation.

anti-inflammatory, anti-angiogenic, and anti-microbial, and it also

1. Introduction
The high complexity of the ocular anatomy represents a great chal­
lenge in the development of new drugs for the treatment of the ocular
diseases. Over the last two decades, numerous drugs have been devel­
oped for the treatment of multiple ocular pathologies. Although many of
these drugs have demonstrated to be beneficial by reducing the symp­
toms of these disorders, there are still many unresolved technical and
medical problems, or they are not cost efficient. In order to heal some
ocular defects, some therapies composed by a tridimensional meshwork
like amniotic membrane (AM), or tissue derivatives (collagen or fibrin)
have been historically used even though they are sometimes insufficient
(Feng et al., 2014). The AM is used for the treatment of several ocular
pathologies because AM has several properties such as anti-fibrotic,
promotes re-epithelialization making it extremely useful for the treat­
ment of ocular diseases (Fernandes et al., 2005). The AM has been
widely used for the treatment of diverse ocular surface disorders
including persistent epithelial defects, neurotrophic ulcers, pterygium,
and corneal surface reconstruction (Alemañy González and Camacho
Ruaigip, 2006; Fernandes et al., 2005; Liu et al., 2010), but also for the
treatment of retinal diseases like macular hole or age related macular
degeneration (AMD) (Florent et al., 2020; Proença and Magro, 2020;
Rizzo et al., 2020). Nevertheless, the use of AM presents some limita­
tions such as the risk of disease transmission, its limited transparency, its
scarce availability and its high cost (Adds et al., 2001; Rahman et al.,
2009).
One novel alternative for the treatment of these diseases is the use of
the plasma rich in growth factors (PRGF) technology, which is a type of

* Corresponding author. Instituto Eduardo Anitua; c/ Jose Maria Cagigal 19, 01005, Vitoria, Spain.
E-mail address: eduardo@fundacioneduardoanitua.org (E. Anitua).

https://doi.org/10.1016/j.exer.2020.108402
Received 25 September 2020; Received in revised form 10 November 2020; Accepted 11 December 2020
Available online 14 December 2020
0014-4835/© 2020 Elsevier Ltd. All rights reserved.
E. Anitua et al.
Abbreviations
PRGF plasma rich in growth factors
mPRGF PRGF membrane
HK keratocytes
HConF conjunctival fibroblasts
AM amniotic membrane
AMD age related macular degeneration
EGF epidermal growth factor
PDGF-AB platelet-derived growth factor-AB
TGF-β1 transforming growth factor β1
FGFb basic fibroblast growth factor
platelet-rich plasma obtained under a standardized and optimized pro­
tocol for tissue regeneration (Anitua, 1999). This technology exhibits a
wide versatility offering several autologous formulations, including an
injectable formulation, growth factor enriched-eye drops, a 3D fibrin
scaffold, and a biomimetic and elastic fibrin membrane (mPRGF) (Ani­
tua et al., 2004, 2015b, 2016a). The mPRGF properties are similar to
those of AM, such its capability to induce tissue regeneration, its
bacteriostatic activity, its anti-inflammatory, and its antifibrotic poten­
tial (Anitua et al., 2012, 2013a, 2015a, 2016b). Furthermore, mPRGF
has been recently used in several clinical studies in the ophthalmic field
with successful results (Arias et al., 2019; Rodríguez-Agirretxe et al.,
2018; Sánchez-Ávila et al., 2019; Sanchez-Avila et al., 2018b).
However, several inflammatory processes including complement
activation, pro-inflammatory cytokine release, and oxidative damage
are involved in the development of various ocular pathologies like AMD,
diabetic retinopathy, or dry eye disease in patients with Sjögren syn­
drome or graft versus host disease (Mima et al., 2012; Munir and Ayl­
ward, 2017; Parmeggiani et al., 2013; Pflugfelder et al., 1999). In these
cases, a reduction of some immunogenic factors in PRGF formulations
could improve the clinical outcomes of these treatments. In a previous
study, our group developed a PRGF formulation based on eye drops with
a reduced proinflammatory content (immunosafe PRGF eye drops)
(Anitua et al., 2014). These eye drops have been successfully used for the
treatment of some ocular diseases in which an immunological compo­
nent is involved in their development (Sanchez-Avila et al., 2017,
2018a).
In the present work, we propose a new type of plasma rich in growth
factors membrane (mPRGF) for patients with autoimmune diseases or
suffering from ocular diseases with an associated inflammatory
component. A temperature of 56 ◦ C was used to obtain the new mPRGF.
Our hypothesis holds that the heat-inactivation of the mPRGF will
disable the complement system without reducing neither its protein
content nor its biological characteristics. Assuming this, a new heat-
inactivated PRGF membrane has been developed and characterized.
The concentration of several growth factors, proteins, immunoglobulins
and complement activity of this new mPRGF have been analyzed. In
addition, the effects of the heat-inactivated membrane on cell prolifer­
ation of different ocular surface cells have been evaluated. The
morphological characteristics and the degradation capability of the new
mPRGF were also evaluated.
2. Materials and methods
2.1. Preparation of personalized PRGF membrane
Blood from three healthy donors was collected into 9-mL tubes with
3.8% (wt/v) sodium citrate, after having signed an informed consent.
The study was performed following the principles of the Declaration of
Helsinki. Whole column of plasma rich in growth factors was collected
after centrifugation avoiding the buffy coat that contains the leukocytes
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Experimental Eye Research 203 (2021) 108402
VEGF vascular endothelial growth factor
END endostatin
TSP-1 thrombospondin-1
ANG-1 angiopoietin-1
ELISA enzyme-linked immunosorbent assay
IgE immunoglobulin E
PETIA particle-enhanced turbidimetric immunoassay
DMEM Dulbecco’s modified Eagle’s medium
H-E: Hematoxilin&Eosin
SEM scanning electron microscopy
tPA tissue plasminogen activator
using Endoret ophthalmology kit (BTI Biotechnology Institute, S.L.,
Vitoria, Álava, Spain). Platelets and leukocytes counts were performed
with a hematology analyzer (Micros 60, Horiba ABX, Montpelier,
France). The whole PRGF volume was activated with Endoret activator
(BTI Biotechnology Institute, S.L., Vitoria, Álava, Spain). Then, the
volume of the obtained PRGF from each donor was divided in two
groups: 1) one part was used to obtain the conventional PRGF mem­
brane (mPRGF): The PRGF was activated with calcium chloride and
incubated at 37 ◦ C for 30 min (used as a control); 2) the other part was
used to obtain the immunosafe PRGF membrane (is-mPRGF): PRGF was
activated with calcium chloride and incubated for 30 min at 56 ◦ C.
2.2. Proteins and growth factor measurements
2.2.1. Growth factor levels
To analyze the effect of heat inactivation on mPRGF, the concen­
tration of several growth factors involved in ocular wound healing, such
as, epidermal growth factor (EGF), platelet-derived growth factor-AB
(PDGF-AB), transforming growth factor β1-(TGF-β1), vascular endo­
thelial growth factor (VEGF), endostatin (END), thrombospondin-1
(TSP-1) and angiopoietin-1 (ANG-1) were measured using commer­
cially available Quantikine colorimetric sandwich enzyme-linked
immunosorbent assay (ELISA) kits according to the manufacturer’s in­
structions (R&D Systems, Minneapolis, MN). The immunoglobulin E
(IgE) was analyzed by a commercially available particle-enhanced
turbidimetric immunoassay (PETIA) obtained from Biokit (Quantex
IgE kit, Barcelona, Spain). PETIA technique was performed according to
the user manual.
2.2.2. Complement activity analysis
Total classical complement activity was measured using a commer­
cial CH50 assay kit from Wako Chemicals (Autokit CH50, Richmond,
VA) based on the technique of liposome immunoassay developed by
Yamamoto et al. (1995). CH50 assay was carried out according to the
protocol handbook.
2.2.3. Growth factors release kinetics
The release kinetics of several growth factors from the heat treated
mPRGF formulation was determined by ELISA assays. Briefly, 2 mL of
PRGF obtained from each donor were added into 6-well plates to obtain
the different PRGF membranes (mPRGF and is-mPRGF). After that,
Dulbecco’s modified Eagle’s medium (DMEM)/F12 media (Gibco-Invi­
trogen, Grand Island, NY, USA) was added and samples were maintained
at 37 ◦ C in a humidified 5% CO2 atmosphere. After 1 h, 1, 3 and 7 days of
incubation, medium was collected and centrifuged at 500 g for 10 min
and the obtained supernatant was aliquoted and stored at − 80 ◦ C until
use. Several growth factors such as EGF, PDGF-AB, TGF-β1, basic
fibroblast growth factor (FGFb), VEGF and END (R&D System) were
evaluated according to the manufacturer’s instructions.
E. Anitua et al.
2.3. Characterization
2.3.1. Histological analysis
Each mPRGF developed in the present study was also histologically
analyzed. Briefly, each mPRGF were fixed in 10% formalin for 24 h.
Then, they were dehydrated and embedded in paraffin. Afterwards, 6
μm sections were cut from the blocks and the different samples were
stained with Hematoxilin&Eosin (Sigma-Aldrich, St. Louis, MO). Several
photographs were captured with different augmentations with a digital
camera coupled to a Leica DM IRB optical microscope (Leica Micro­
systems, Wetzlar, Germany). The platelets and fibrin fiber distribution
were analyzed across the width of each mPRGF obtained in the study.
2.3.2. Fluorescence staining of fibrin membrane
The morphology and distribution of fibrin fibers were also evaluated
by immunofluorescence techniques. Briefly, fluorescent fibrinogen
(Invitrogen, Carlsbad, CA) at a concentration of 125 μg/mL was added to
the PRGF samples obtained from each donor, thus achieving the
different fibrin membranes included in the study (mPRGF and is-
mPRGF). The different mPRGF were subsequently analyzed under a
confocal microscope (Leica LCS SP2 AOBS, Leica Microsystems, Wetzlar,
Germany).
2.3.3. Scanning electron microscopy
Scanning electron microscopy (SEM) was employed to evaluate the
3D structure of the different mPRGF obtained in the study. Samples were
fixed with 2.5% glutaraldehyde and post-fixed with osmium tetroxide
(1% OsO4 in 0.1M cacodylate) and finally dehydrated through
ascending alcohol concentrations. Thereafter, mPRGFs were subjected
to critical point drying (Autosamdri 814. Tousimis, Rockville), gold
sputter coated and imaged using an electron microscope (S-4800;
Hitachi, Japan).
2.3.4. In vitro degradation study
The degradation of the different mPRGF developed in the present
work was carried out upon exposure to tissue plasminogen activator
(tPA) over a period of 7 days. The conversion of plasminogen to plasmin
is catalyzed by tPA, which finally induces the degradation of plasmatic
fibrin. Briefly, the different mPRGF were prepared as described above
and incubated for 60 min at 37 ◦ C to allow the complete fibrin forma­
tion. Samples were then immersed into PRGF for 24 h at 37 ◦ C to allow a
complete fibrin contraction. These were then weighed to obtain the
initial weight (W0) and transferred into PRGF containing tPA (0.25 μg/
ml, AbCam Cambridge, UK) at 37 ◦ C. Weight loss was recorded at
different time points (1, 2, 3, 6 and 7 days). Finally, the percentage of
mass remaining of each mPRGF was calculated using the following
equation.
Mass remaining % = (Wd/W0) x 100
where W0 was the initial weight of each mPRGF and Wd is the weight of
the same mPRGF at each study time.
2.4. In vitro cell studies
2.4.1. Cell culture
Two different types of ocular surface cells including primary human
keratocytes (termed HK) and conjunctival fibroblasts (termed HConF)
(ScienCell Research Laboratories, San Diego, CA) were used to evaluate
the biological activity of both types of mPRGF. HK and HConF cells were
cultured in Fibroblast medium supplemented with Fibroblast Growth
Supplement (ScienCell Research Laboratories, San Diego, CA), 2% fetal
bovine serum and antibiotics in a humidified atmosphere of 5% CO2 at
37 ◦ C following the manufacturer’s instructions.
After confluence, cells were detached with animal origin-free
trypsin-like enzyme (TrypLE Select, Gibco-Invitrogen, Grand Island,
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Experimental Eye Research 203 (2021) 108402
NY, USA). Cell viability was assessed by trypan blue dye exclusion. Cells
in passage 3–5 were used in the assays.
2.4.2. Proliferation assay
The biological activity of each mPRGF was determined by evaluation
of HK and HConF proliferation. Briefly, the different PRGF membranes
(mPRGF and is-mPRF) obtained from each donor were squeezed through
a membrane former (BTI Biotechnology Institute, Vitoria, Spain) and the
resulting supernatant was filtered, aliquoted and stored at − 80 ◦ C until
use. The ocular surface cells (HK and HConF) were seeded in a 96-well
dark-bottom plate at a density of 10,000 cells per cm2 in serum-free
medium supplemented with 20% (v/v) of the different supernatant
obtained from each mPRGF mentioned above. The study period was 72
h. Cell proliferation was assessed by Cyquant cell proliferation assay
(Molecular Probes-Life technologies) according to the manufacturer’s
protocol.
2.5. Statistical analysis
Data were expressed as mean values and standard deviations. Paired
t-test or Wilcoxon test were used to analyze the differences between the
mPRGF obtained in the present study for the different variables studied.
Statistical significance level was set on p < 0.05. SPSS v15.0 for Win­
dows statistical software package (SPSS Inc., Chicago, IL, USA) was used
for all statistical analysis.
3. Results
The mean platelet enrichment of the PRGF preparations was 2.15-
fold over the baseline concentration of platelets in whole blood. The
PRGF platelet concentration obtained from the three donors ranged
from 373 to 560 × 103 plts/μL. None of the preparations contained
detectable levels of leucocytes. The morphological appearance of the
different mPRGF included in the present study were similar (Fig. 1). The
fibrin clot of the control group had begun to retract itself after 30 min of
incubation at 37 ◦ C in comparison to the is-mPRGF, which maintained
its initial shape at this incubation time.
3.1. Proteins and growth factor measurements
3.1.1. Growth factor levels
The concentration of several growth factors involved in ocular sur­
face regeneration (EGF, PDGF-AB, TGF-β1 and VEGF) as well as some
involved in the angiogenesis processes (TSP-1, ANG-1 and END) were
analyzed in the control mPRGF (Control) and the heat-treated immu­
nosafe mPRGF (is-mPRGF). The levels of the different growth factors
analyzed are presented in Table 1. The different growth factors involved
in ocular tissue regeneration showed similar levels among the different
mPRGF studied in the present work. However, the concentration of the
Fig. 1. Representative images of the macroscopic appearance of the different
mPRGF analyzed in the present study.
E. Anitua et al.
Table 1
Concentration of the growth factors analyzed in the different mPRGF samples obtained in the study from the three donors (Mean ± standard deviation).
PDGF-AB (ng/mL) EGF (pg/mL) TGF-β1 (ng/mL)
mPRGF 15.7 ± 6.0 379 ± 117 11.3 ± 2.3
is-mPRGF 14.8 ± 6.3 476 ± 154 8.9 ± 1.3
*Significant differences compared with control group.
growth factors related with angiogenesis processes were reduced
significantly (p < 0.05) in immunosafe mPRGF samples regarding the
control mPRGF (Table 1).
3.1.2. Components of immune system
The concentration of IgE and the complement activity were
measured to check the heat inactivation of mPRGF. The concentration of
IgE was significantly reduced (p < 0.05) after heat-inactivation of the
mPRGF (is-mPRGF) with respect to the control mPRGF (Fig. 2).
Heat-treated mPRGF showed a significant reduction (p < 0.05) in the
complement activity (CH50) in comparison to the control group (Fig. 2).
In fact, since complement activity values in the heat-inactivated mPRGF
were below the detection limits of the assay, it could be considered a
complete inactivation of the complement system.
3.1.3. Release kinetics of growth factors
The results of the release kinetics assay show that most of the growth
factors and proteins analyzed reach their maximum release level on day
1, and they were subsequently maintained at the same release levels on
the remaining evaluated days (3 and 7 days) (Fig. 3). However, in the
case of FGFb a peak release of the growth factor is observed at day 1 in
the control group (mPRGF), while in the case of immunosafe mPRGF
there was a reduction in the release of this factor after 1h from its
collection. In contrast, in the case of TGF-β1, there was a continuous
increase in the release of the TGF-β1 throughout the different time
points studied.
No significant differences (p > 0.05) were observed in the release
levels of the diverse growth factors and proteins analyzed among the
mPRGF samples obtained in the present work at any of the study times
analyzed.
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Experimental Eye Research 203 (2021) 108402
VEGF (pg/mL) END (ng/mL) TSP-1 (μg/mL) ANG-1 (μg/mL)
130 ± 113 124 ± 28 31.4 ± 10.3 32.7 ± 9.7
178 ± 147 3 ± 1* 12.9 ± 1.7* 13.1 ± 3.5*
3.2. Morphological characterization of the different mPRGF
The histological study (Fig. 4) showed that the control fibrin mem­
branes (mPRGF) are more intensely stained than the immunosafe
mPRGF. One possible reason for this fact is a greater presence of fibrin
fibers and platelets in the different areas of the control membranes
(Fig. 4A, C, 4E and 4G) regarding is-mPRGF (Fig. 4B, D, 4F and 4H).
Fibrinogen fluorescent staining corroborated the results observed
with the histological techniques. The control membrane group con­
tained higher number of both fibrin fibers and platelets than those
observed in immunosafe mPRGF (Fig. 4I–J). On the contrary, is-mPRGF
had smaller number of fibrin fibers and platelets and these were
distributed in a more dispersed way and without the formation of
platelet aggregates along the whole PRGF membrane.
Scanning electron microscopy analysis showed that there were no
significant differences in the morphology of the fibrin fibers that made
up the different mPRGF developed in the study (Fig. 4K–L). Moreover,
the results obtained with previous techniques were also corroborated
with the SEM technique, revealing a greater amount of fibrin fibers in
the control group (Fig. 4K) with respect to the is-mPRGF (Fig. 4L).
3.3. Fibrin degradation study
To evaluate the potential of the new is-mPRGF to be degraded upon
exposure to enzymes found in vivo, they were incubated with tPA, the
enzyme that catalyzes the conversion of plasminogen to plasmin (Fig. 5).
The weight loss of both types of PRGF membranes (mPRGF and is-
mPRGF) was reduced along the study period until they were
completely degraded after 7 days of incubation with tPA. No significant
differences (p > 0.05) were found between the different mPRGF at any
study time.
Fig. 2. Complement activity and IgE detec­
tion in non-inactivated and heat-inactivated
PRGF membrane samples. (A) The levels of
immunoglobulin E were significantly
reduced in mPRGF after heat-inactivation
(is-mPRGF) in comparison to the non-
inactivated PRGF membrane (mPRGF). (B)
Complement activity was completely
reduced in the immunosafe mPRGF (is-
mPRGF) after heat-treatment at 56 ◦ C for 30
min *p < 0.05. D1: donor 1; D2: donor 2; D3:
donor 3. Means are represented by a bar in
each treatment group.
E. Anitua et al.
Fig. 3. Release kinetics of different growth factors (PDGF-AB, EGF, TGF-β1, FGFb, VEGF and END) from the different mPRGF incubated at 37 ◦ C for 1 h and 1, 3 and
7 days. No differences were found between the immunosafe mPRGF and the control mPRGF in any of the growth factors analyzed at any of the study time points.
3.4. Biological activity of heat treated mPRGF
The biological activity was measured as proliferative potential of
mPRGF samples developed in the study in HK and HConF cells. Both cell
types showed no significant differences in their proliferation levels after
treatment with the different mPRGF samples obtained along the study
(Fig. 6).
4. Discussion
The role of autoantibodies has been suggested in the development of
several ocular autoimmune diseases, such as rheumatoid arthritis, sys­
temic lupus erythematosus, Sjögren’s syndrome and graft versus host
disease (Mohsenin and Huang, 2012; Perez et al., 2013). In addition, the
complement activation has been involved in the progress of these ocular
autoimmune diseases through the binding to Fc receptors of the auto­
antibodies (Daha et al., 2011; Nguyen et al., 2007). Furthermore,
complement activation has been suggested to be involved in the devel­
opment of several ocular diseases where diverse inflammatory processes
are implicated in their pathogenesis including AMD (Kauppinen et al.,
2016; Mateos et al., 2015), glaucoma (Reinehr et al., 2016; Williams and
Dellinger, 2016), and diabetic retinopathy (Huang et al., 2018; Ras­
mussen et al., 2018). In the present study, we described a complete
inactivation of complement system after heating of PRGF membranes at
56 ◦ C for 30 min, thus preventing the translation of immune disorders to
ocular tissues by using PRGF fibrin membranes for the treatment of
diverse ocular diseases. It should be highlighted that the most significant
achievement of this study is related to the reduction or complete
denaturalization of some components of the immune system from the
PRGF membrane after the heating process. In fact, a reduction of more
than 65% in the content of IgE was obtained in the heat-inactivated
mPRGF. These results suggest that mPRGF inactivation could be effec­
tive to treat ocular disorders in patients with allergic diseases (McGill
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Experimental Eye Research 203 (2021) 108402
et al., 1998). In those cases where we want to reduce the concentration
of IgE, the mPRGF treatment time could be increased at 56 ◦ C until 60
min without reducing the concentration of proteins that could be
involved in tissue regeneration (Anitua et al., 2014).
This study shows the development and characterization of a new
type of plasma rich in growth factors membrane for the treatment of
ocular disorders in patients suffering from autoimmune diseases or pa­
thologies with implication of the complement activation. The protein
and growth factors content of the heat-inactivated mPRGF was analyzed
in comparison with the non-inactivated mPRGF. Their biological po­
tential was also determined in two different cell types: keratocytes (HK)
and conjunctival fibroblasts (HConF). In addition, morphological and
physics features of the inactivated mPRGF were evaluated in contrast to
the control mPRGF (non-inactivated).
The results show that most of the measured growth factors related to
tissue regeneration including PDGF, TGF-β1, EGF and VEGF were
maintained at the same concentrations in the immunosafe mPRGF, thus
confirming that the composition of the main growth factors remained
unchanged after 30 min of heat-treatment. The preservation of the main
protein factors related to the tissue regeneration was reflected in the
biological activity of the different mPRGF developed in the study, with
no change in the proliferative rate of the ocular surface cells (HK and
HConF) after treatment with the immunosafe membrane compared to
the control membrane. On the other hand, some growth factors related
to angiogenesis processes like ANG-1 with an angiogenic effect (Suri
et al., 1996) and END and TSP-1 with an anti-angiogenic effect (Irue­
la-Arispe et al., 1999; O’Reilly et al., 1997) were significantly reduced
after heat treatment of the mPRGF. Several studies have demonstrated
that the different PRGF formulations are composed by a myriad of
proteins and morphogens (Anitua et al., 2015c; Nurden et al., 2008).
Moreover, it is widely known that each stage of the repair process
including angiogenesis is controlled by a wide variety of growth factors
acting locally as regulators of the most basic cell functions, using
E. Anitua et al.
Fig. 4. Morphological characterization of the fibrin clot obtained from the
different mPRGF developed in the study. (A–H) Representative images of the
control mPRGF and the is-mPRGF stained with hematoxylin and eosin (H–E).
The control mPRGF (A) is stained more intensely than the immunosafe mPRGFs
(B). High magnification images show a higher number of fibrin fibers and
platelets in control mPRGF in comparison with is-mPRGF. These results were
corroborated with both the fibrinogen fluorescent staining assay (I and J) and
the scanning electron microscopy analysis (K and L).
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Experimental Eye Research 203 (2021) 108402
Fig. 5. Biodegradation rate of the different mPRGF after incubation with tissue
plasminogen activator for 7 days. No differences were observed in the degra­
dation time between the both types of PRGF membranes (mPRGF and is-
mPRGF) at any study time.
endocrine, paracrine, autocrine and intracrine mechanisms (Werner and
Grose, 2003). In this sense, future studies should be necessary to analyze
the effect of this protein reduction related to the angiogenic process in
the immunosafe mPRGF.
Ayache et al. showed that a large range of proteins including
fibrinogen were partially denatured after heat inactivation of plasma at
56 ◦ C for 1 h (Ayache et al., 2006). Additionally, it has been demon­
strated that fibrinogen concentration is directly correlated with fibrin
stiffness and clot density and it is inversely correlated with clot degra­
dation rate (Collet et al., 2000; Weisel, 2005). Furthermore, the fibrin­
ogen, thrombin and FXIIIa concentrations define the characteristics of
the fibrin clot such as clot microstructure (pore size, density), stiffness
and resistance to fibrinolysis. In addition, it has been demonstrated that
these parameters also influence the release kinetics of the different
growth factors contained in the fibrin clot (Catelas et al., 2008; Zhou
et al., 2011). On the other hand, several studies have evaluated the ef­
fects of fibrin composition on the growth of different cell types like fi­
broblasts, keratinocytes, and mesenchymal stem cells, showing that a
fibrin matrix with an intermediate or soft stiffness favors the growth of
all these cell types (Bensaïd et al., 2003; Cox et al., 2004; Sese et al.,
2011). This is especially important for the wound healing process
because a lower fibrin clot stiffness seems to be beneficial for tissue
repair, however, this type of matrix could be degraded rapidly and
therefore, it would not be suitable for the sustained release of growth
factors over long time periods (Anitua et al., 2013b; Heher et al., 2018).
The morphological evaluation of the PRGF membranes obtained in the
present study shows that the fibrin clot inactivated by heat treatment
(is-mPRGF) seems to contain less amount of fibrin fibers than the
non-inactivated fibrin membrane (control). This could result in an
augmentation in the release kinetics of the growth factors contained in
the fibrin clot of the immunosafe mPRGF, as well as a faster fibrin
degradation. However, the present study shows that the different growth
factors analyzed were similarly released from the heat-treated mPRGF as
from the non-heat treated mPRGF. In addition, no differences were
found between the immunosafe membrane and the control mPRGF in
the degradation time after incubation with tPA.
5. Conclusions
In summary, this study describes the development and character­
ization of new type of heat-inactivated PRGF membrane. This immu­
nosafe mPRGF reduces drastically the content of IgE and complement
activity while preserving the content of most of the proteins and mor­
phogens involved in ocular wound healing. Furthermore, the morpho­
logical and physical features of the immunosafe mPRGF were also
E. Anitua et al.
Fig. 6. Biological activity of corneal keratocytes (HK) and conjunctival fibroblasts (HConF) after culture with control mPRGF or with heat-inactivated mPRGF (is-
mPRGF). No significant differences were observed in the proliferation activity of the HK and HConF cells after being treated with the different study samples. D1:
donor 1; D2: donor 2; D3: donor 3. Means are represented by a bar in each treatment group.
conserved after heat-inactivation. Although PRGF eye drops has been
extensively used in patients with autoimmune mediated dry eye and
ocular surface inflammatory conditions with no symptom exacerbation,
and thus, clinical studies are needed to evaluate the efficacy of this new
formulation, this is the first time that a heat-inactivation treatment of a
platelet-rich plasma membrane is suggested to treat ocular diseases in
patients where the immune system is involved in the etiology of their
disease.
Declaration of competing interest
The authors declare the following competing financial interest(s): E.
A. is the Scientific Director and M.F. and F.M. are scientists at BTI
Biotechnology Institute, a company that investigates in the fields of oral
implantology and PRGF-Endoret technology.
Acknowledgements
This study is based on data generated in the BIORETINA project (nº
ZL-2017/00303 y ZL-2018/00154), funded by the HAZITEK program of
the Department of Economic Development and Infrastructure of the
Basque Country Government.
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