Acta Biomaterialia 177 (2024) 132–147

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio

Full length article

Characterization of the molecular composition and in vitro

regenerative capacity of platelet-based bioproducts and related
subfractions
Andrea Acebes-Huerta a,b, Patricia Martínez-Botía a, Graciela Carbajo-Argüelles a,
Judit Fernández-Fuertes a,c,d, María Carmen Muñoz-Turrillas a,e, Ana María Ojea-Pérez f,
Antonio López-Vázquez g, Johannes A. Eble h, Laura Gutiérrez a,b,∗
a
Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain
b
Department of Medicine, University of Oviedo, Spain
c
Department of Orthopedics and Trauma Surgery, Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain
d
Department of Surgery and Medical Surgical Specialties, University of Oviedo, Spain
e
Centro Regional de Transfusión de Toledo-Guadalajara, Spain
f
Centro Comunitario de Sangre y Tejidos de Asturias, Oviedo, Spain
g
Department of Immunology, Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain
h
Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. How-
Received 13 March 2023 ever, it is very difficult to establish the real clinical benefits of these therapies, as the lack of charac-
Revised 29 December 2023
terization and detailed production methods of platelet-based bioproducts persists in the literature and
Accepted 19 January 2024
precludes cross-study comparisons. We characterized the molecular composition and in vitro regenerative
Available online 3 February 2024
capacity of platelet-rich plasma (PRP) produced in a closed-system. Furthermore, we performed a parallel
Keywords: characterization on different PRP subfractions (plasma and plasma-free platelet lysate), identifying that
Platelets the fractions containing platelet-derived cargo exert the most potent regenerative capacity. This observa-
Platelet rich plasma (PRP) tion led us to develop a method to obtain a platelet secretome highly enriched in growth factors, free of
Plasma plasma and cellular components (PCT/IB2022/057936), with the aim of establishing a superior bioprod-
Platelet secretome uct. The molecular characterization of secretomes revealed agonist-dependent differences, which corre-
Bioactive factors
lates with beneficial grades of regenerative capacity. Importantly, secretomes showed general superiority
Growth factors
Wound healing
to PRP in vitro. We discuss the variables influencing the bioproduct quality (inter-donor variation, platelet
Regenerative medicine source and processing methods). Finally, we propose that the characteristics of secretomes circumvents
certain limitations of PRP (autologous vs allogeneic), and envision that optimizing post-processing proto-
cols (nanoencapsulation, lyophilization), would allow their clinical application even beyond regenerative
medicine.

Statement of significance

The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. How-
ever, it is very difficult to establish the real clinical benefits of these therapies, or to improve/personalize
them, as the lack of characterization of the bioproducts and their production methods is a constant in
the literature, reason that precludes cross-study comparisons. In the present manuscript, we provide a
comprehensive molecular and functional characterization of platelet-based bioproducts and subfractions,
including platelet rich plasma, plasma fractions and platelet secretomes produced with a methodology
developed by our group. Our results show that the molecular composition of each fraction correlates

∗
Corresponding author at: Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Edificio FINBA, Platelet Research Lab NO-F15, Avenida del Hospital Univer-
sitario S/N, 33011 Oviedo, Spain.
E-mail address: gutierrezglaura@uniovi.es (L. Gutiérrez).

https://doi.org/10.1016/j.actbio.2024.01.029
1742-7061/© 2024 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al.
with its regenerative capacity in vitro. Thus, a rigorous characterization of platelet-derived bioproducts
will potentially allow universal use, customizing and new applications.
© 2024 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc.
1. Introduction
Platelets are circulating anucleate blood components (2-4 μm
in diameter) and key players in maintaining the body hemosta-
sis [1]. Noteworthy, they also participate in other physiological and
pathological processes beyond hemostasis and thrombosis, such as
immunomodulation, lymph and blood vessel separation during de-
velopment, angiogenesis and tumor metastasis [2–6]. Hemostatic
platelet function and communication with the coagulation cas-
cade is regulated by receptor-mediated signaling networks leading
to platelet adhesion, secretion and aggregation [7]. As a result of
vessel damage, the exposed subendothelial and secreted adhesive
proteins (i.e., collagen and von Willebrand factor), are recognized
by platelets through their receptors, which induces their activa-
tion and adhesion to the site of injury, cytoskeletal rearrangements
(filopodia and lamellipodia formation), integrin activation (essen-
tial for platelet aggregation), and secretion of the platelet granule
cargo or secretome [8–10].
The platelet secretome consists of various sorts of molecules,
which will assure the amplification of the platelet activation sig-
nal, the recruitment of circulating blood cells such as leukocytes
and fibroblasts to the site of injury, and the repair of the injured
vessel. These molecules are packed in three major types of gran-
ules, which differ in content, biogenesis, trafficking, and exocyto-
sis [11]. The alpha granules (α -granules) are the most abundant
(approximately 50-80 per platelet) and contain the proteins that
comprise the secretome core, i.e., membrane-associated receptors
(such as α IIbβ 3 and P-selectin) and soluble cargo (growth fac-
tors, cytokines, anti- and pro-angiogenic factors, proteases, etc.)
that participate in a wide variety of biological processes, includ-
ing hemostasis, inflammation, cancer and wound healing [10,12-
14]. Some of these molecules are synthesized and packaged into α -
granules in the megakaryocytes prior to platelet formation, while
others, such as fibrinogen, clotting factor V and VEGF (among oth-
ers) are (also) endocytosed and incorporated into α -granules ei-
ther by megakaryocytes or by circulating platelets [15–19]. Dense
granules (δ -granules) are the second most abundant granule type
found in platelets, and contain small molecules such as serotonin,
calcium and ADP, which serve to amplify platelet activation. Lastly,
platelets also contain lysosomes (1-3 per platelet), rich in acid
hydrolases (cathepsins, hexosaminidase, β -galactosidase, arylsulfa-
tase, β -glucuronidase and acid phosphatase) [11]. Thus, the platelet
secretome plays a central role in the regulation of hemostasis
by driving cell-to-cell interactions and tissue regeneration during
wound healing. Additionally, it recently emerged that both the
qualitative and quantitative composition of the secretome, as well
as its release dynamics, may be more complex and orchestrated
than anticipated [10,14,20,21].
In recent years, the specific healing property of platelets and
their cargo has been used as rationale for the development of
platelet-based bioproducts as adjuvant and stand-alone treatment
in advanced cell therapy and regenerative medicine. Platelet-rich
plasma (PRP) is the most popular platelet-based bioproduct and
its therapeutic application constitutes a relatively new approach
in regenerative medicine, with clinical benefits in a wide range
of medical fields, such as odontology and maxillofacial surgery,
ophthalmology, traumatology, sports and aesthetics, and veterinary
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Acta Biomaterialia 177 (2024) 132–147
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
medicine, among others [22–24]. However, the therapeutic out-
come of the use of PRP remains highly controversial due to a lack
of consensus on PRP preparation, post-production processing and
application methods [25]. As a consequence, the quality of PRPs
varies considerably amongst studies in terms of platelet number,
presence or absence of white/red blood cells, and molecular com-
position, which also lacks characterization [26]. This adds to the
fact that in some studies, PRP is applied either fresh (with living
cellular components), or after freezing or activation which induces
platelet lysis or platelet granule cargo release. Furthermore, in PRP-
like bioproducts, the total platelet proteome is contained within a
mixture of plasma proteins, of which the actual bio-active compo-
nents (i.e., those contained in platelet granules) represent a rather
small sub-fraction of the product. Another important issue con-
cerns the regulatory aspects. In most territories, the application of
PRP is only allowed in an autologous manner. This requires that the
patient must schedule a blood donation to prepare the PRP prior
application. However, it has been reported that certain pathological
conditions might influence platelet quality and reactivity, and an
autologous PRP might not always be the most suitable option for
therapy, since it could result in uncertain clinical outcomes, due to
inter-donor as well as pathological- or subclinical-dependent vari-
ations [27–29].
We propose that a better understanding of the nature and the
active components of platelet-based bioproducts is crucial to opti-
mize and standardize production methods as to improve the clini-
cal benefit of the therapy.
In the present manuscript we characterize the molecular com-
position of PRPs from different donors produced in a standardized
closed-system using the infrastructure of a certified blood bank
and processed by snap-freezing to allow platelet lysis [30] and as-
sociate it to their in vitro regenerative capacity. Furthermore, we
delve into the contribution of different PRP subfractions (plasma
and plasma-free platelet lysate), that have been individually char-
acterized at the molecular level, to the observed regenerative ca-
pacity. The observation that the bioproduct and subfractions that
exert the most potent regenerative capacity are those contain-
ing platelet-derived cargo, led us to develop a method to obtain
a platelet secretome enriched bioproduct (PLT-S), free of plasma
and cellular components (PCT/IB2022/057936). We deepen into the
molecular and functional characterization of PLT-Ss and compare
them with PRP and PRP subfractions. We further discuss the vari-
ables influencing the final quality of PLT-Ss (i.e., inter-donor vari-
ation, platelet biological source and processing) and the intrinsic
characteristics that would allow further developments of platelet-
based bioproducts with clinical therapeutic purposes even beyond
regenerative medicine.
2. Material and methods
The study did not require recruitment of patients or healthy
donors, as we used interrupted blood donations or discard material
from the Local Blood Bank (Centro Comunitario de Sangre y Tejidos
de Asturias – CCSTA). Therefore, informed consent or approval by
the ethics committee was not required per se in this setting. The
line of research to which this study belongs has obtained approval
by the Local Medical Ethical Committee (#33/19, Hospital Universi-
tario Central de Asturias, Oviedo, Spain). The study was conducted
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al.
according to the Declaration of Helsinki, and all materials and sam-
ples used were anonymized.
2.1. Production of PRP and PRP-derived subfractions
Platelet-rich plasma (PRP) was obtained from interrupted blood
donations (n=14). The fractionation was performed using the
equipment of a certified blood bank, as previously described [30].
Briefly, the plasma fraction containing platelets was separated from
the fraction containing red blood cells after centrifugation (1130
rpm for 5 minutes, without brake) with a press Compomat G5 sys-
tem (Fresenius-Kabi) into a separate bag. A second centrifugation
(2500 rpm for 12 minutes, brake 4) was performed to obtain an
enriched PRP fraction, after removal of the upper phase, the so-
called platelet poor plasma (PPP), into a separate bag. The PRP
was then distributed into 3 pediatric bags (each of them contain-
ing approximately 10 mL), and stored at −40°C. When indicated,
a complete blood count was performed prior freezing (Sysmex
XN-10/XN-20, Hematology Analyzer). For experiments and molec-
ular characterization, PRP samples were subject to 2 additional
freezing-thawing cycles to assure complete platelet lysis (-20°C -
room temperature) [31] and then, centrifuged at 10k rpm for 2
minutes, to discard cell debris. The supernatants were collected
through a 0.22 μm pore filter, aliquoted and stored at -80°C un-
til use (see Fig. 1A).
To prepare PRP-derived subfractions, we used PRP produced
as described above from 4 different donors. We kept samples of
the original PRPs and respective PPPs, and further processed the
remaining PRP fraction by centrifugation (40 0 0 rpm, 5 minutes).
The supernatant was collected and tentatively named PRP Plasma,
and the pelleted platelets were resuspended in the same origi-
nal volume of HEPES Glucose buffer (132 mM NaCl, 6 mM KCl, 1
mM MgSO4 , 1.2 mM KH2 PO4 , 20mM HEPES, pH 7.4, containing 5
mM D-glucose) to produce Plasma-free Platelet Lysate (PFP-L) (see
Fig. 1B). The PPP and PRP Plasma fractions were centrifuged at 10k
rpm for 2 minutes, to discard cell debris, and supernatants were
collected through a 0.22 μm pore filter and kept frozen in aliquots
at -80°C until further use. The PRP and PFP-L fractions were sub-
ject to 2 freezing-thawing cycles (-20°C – room temperature), and
then, centrifuged at 10k rpm for 2 minutes to eliminate cell debris.
Then, the supernatants were collected through a 0.22 μm pore fil-
ter, aliquoted and stored frozen at -80°C until further use.
2.2. Platelet secretome (PLT-S) production
The method to obtain PLT-S is protected by international
patent application (PCT/IB2022/057936). In brief, we prepared PLT-
S from two different biological sources: interrupted blood dona-
tions (whole blood -PLT-SWB -; n=4) and closed-system PRP (PLT-
SPRP ; n=4), (see Fig. 1C). We first collected the PRP fraction from
whole blood samples by differential centrifugation (10 0 0 rpm for
15 minutes, without brake). We counted the platelets in the PRP
fraction from whole blood and in closed-system PRP samples us-
ing an automated hematology analyzer (Sysmex XN-10/XN-20). We
washed the platelets by centrifuging at 40 0 0 rpm for 5 min-
utes, and resuspended them in HEPES Glucose Buffer to a con-
centration of 2 × 108 platelets/mL. Subsequently, platelets were
activated with the following agonists: TRAP6 (100 μM; BACHEM
H-8365.0 0 05), convulxin (0.625 ng/ml; ENZO Life Sciences ALX-
350-100-C050), phorbol 12-myristate 13-acetate (PMA; 100 ng/mL;
Sigma-Aldrich P8139-5MG), aggretin A (6.53 nM), ristocetin (0.5
mg/mL; Sigma-Aldrich R7752-100MG) or collagen (30 μg/mL and
90 μg/mL; Sigma-Aldrich C4243-20ML), under constant stirring
during 5 minutes, at 37°C, using a thermomixer (Mixing Block MB-
102; BIOER). We did not use platelet aggregation inhibitors such as
apyrase and prostaglandin E2 (PGE2), and did not stop the reaction
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Acta Biomaterialia 177 (2024) 132–147
by incubating the platelets on ice. To collect the PLT-Ss, the differ-
ent platelet-activation reactions were immediately centrifuged at
40 0 0 rpm for 5 minutes. The supernatant was transferred to a new
tube, and centrifuged at 10k rpm for 2 minutes to eliminate cell
debris. Supernatants were collected through a 0.22 μm pore filter,
aliquoted and stored frozen at -80°C until further use.
Alternatively, we prepared secretomes from reactions contain-
ing 6 × 108 platelets/mL, to match the average platelet concentra-
tion of closed system PRPs and named them Supra PLT-SPRP . The bio-
logical source used for the preparation of Supra PLT-SPRP was closed-
system PRP (Fig. 1C).
2.3. Quality control of secretomes: flow cytometry analysis of platelet
degranulation
In order to analyze platelet degranulation capacity upon stimu-
lation, platelets were incubated 10 minutes at room temperature
with a panel of surface markers (CD9-Alexa Fluor 647; CD62P-
PE; CD63-PECy7), before and after stimulation as described above
(section 2.2, PLT-S production). After fixation with formaldehyde
to a final concentration of 0.5%, samples were analyzed by flow
cytometry. Platelets were gated based on forward/side scatter and
CD9 positivity. The cut-off was set on unstimulated samples, and
the increase of CD62P and CD63 expression were measured. All an-
tibodies were purchased from BD Biosciences. Data were acquired
on a FACS Aria IITM flow cytometer (BD Biosciences) and analyzed
using FlowJo software (Tree Star, Ashland, OR, USA).
2.4. Production of platelet-count customized PRP and PRP-derived
subfractions
Since the PLT-Ss were prepared from reactions containing
2 × 108 platelets/mL, in some comparison studies we used PRP
and PFP-L produced with the same number of platelets, namely
Custom PRP and Custom PFP-L (Fig. 1D). In brief, we counted the num-
ber of platelets in closed-system PRP samples prepared as de-
scribed above, and resuspended the desired number of platelets
after centrifugation at 40 0 0 rpm for 5 minutes, with either
PRP Plasma from the same sample (Custom PRP) or HEPES Glucose
Buffer (Custom PFP-L). After 2 freezing-thawing cycles (-20°C – room
temperature), samples were centrifuged at 10k rpm for 2 min-
utes to eliminate cell debris. Supernatants were collected through
a 0.22 μm pore filter, aliquoted and stored frozen at -80°C until
further use.
2.5. In vitro scratch wound closure assays
The regenerative potential of the different bioproducts was an-
alyzed in vitro by scratch wound closure assays. To this end, MRC-
5 human fetal lung fibroblast cells were seeded in IbiTreat plates
(μ-Plate 24-well 2-chamber insert Black, Ibidi), at a density of
3.5 × 105 cells/mL (70 μL volume per insert chamber) and incu-
bated to allow cell attachment until reaching 80-90% confluence
(approximately 24 hours) in DMEM supplemented with 1% peni-
cillin/streptomycin (P/S) and 10% Fetal Bovine Serum (FBS). Then,
the silicone insert was carefully removed, leaving a 500 μm cell-
free gap (according to the manufacturerś datasheet). After wash-
ing away detached cells, culture media supplemented with 10% of
platelet-based bioproduct (in the presence or not of 10% FBS, or
in the presence or not of 2 U/mL of heparin to avoid the clot-
ting, as indicated per experiment) was added. Cell-free gaps were
monitored for 48 hours using a time-lapse microscopy Zeiss Axio
Observer Z1 microscope (Carl Zeiss, Germany) equipped with a
‘stage incubator’, keeping the temperature and the CO2 concentra-
tion constant (37°C and 5% CO2 ). Imaging was performed with a
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al. Acta Biomaterialia 177 (2024) 132–147

Fig. 1. Schematic illustration of the production process of the different platelet-derived bioproducts used in the study. A. Closed-system PRP preparation using the infrastruc-
ture of a local blood bank. B-D. Schematic illustration of the processing method to obtain: (B) PRP subfractions, (C) platelet-derived secretomes (PLT-S) and (D) custom-made
PRP and PFP-L alternatives.

135
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al.
Plan-Apochromat 10x/0.45 M27 (dt=2.1mm) objective. Mosaic im-
ages (3 × 3) were taken every 12 hours (AxioCam MRm; Carl
Zeiss). Fields were selected with micrometric precision (in a three-
dimensional manner, axes xyz) using the Tiles tool (z-position from
Tiles Setup) from ZEN 3.2 Blue edition software (Carl Zeiss). The
acquisition software creates automatically the image composite. In
some cases, when the cell confluency is not homogeneous or when
clean areas are identified, the software can make errors in overlap-
ping images; when applying automatic correction for these errors,
a brightness shift appears in the overlap area, without affecting
cell localization and image analysis. The images were subsequently
analyzed using Fiji/ImageJ software (NIH) with the Montpellier
Ressources Imagerie (MRI) “Wound healing tool” macro avail-
able online at: https://github.com/MontpellierRessourcesImagerie/
imagej_macros_and_scripts/wiki/Wound- Healing- Tool. The plugin
analyzes the area of a wound in a cellular tissue. From a stack
of images representing a time-series, it measures the reduction of
the cleared space between the wound edges (cell-free gap). The
percentage of gap closure was calculated and compared between
controls and treatment groups.
2.6. Secretome stability and consumption in vitro
To evaluate the time- and temperature-dependent factor stabil-
ity and consumption of PLT-S in our culture setting, MRC-5 hu-
man fetal lung fibroblast cells were seeded in conventional 24-well
plates or using μ-Plate 24-well 2-chamber insert (Ibidi), at a den-
sity of 3.5 × 105 cells/mL (3.5 × 105 or 0.49 × 105 total cells, re-
spectively) and incubated to allow cell attachment in standard con-
ditions. After this, the medium was replaced by fresh culture me-
dia without serum (DMEM+1% P/S) supplemented with 10% PLT-
S. Supernatants were collected after 24 and 48 hours. As controls,
we took supernatants of cells incubated in the absence of PLT-S,
and of cultures devoid of cells but supplemented with PLT-Ss. The
concentration of a panel of bioactive factors in the collected super-
natants from all conditions was analyzed by Luminex Technology,
as described below (Section 2.7).
2.7. Multiplex immunoassay
Molecular characterization of platelet bioproducts and frac-
tions was performed using high-performance multiplex immunoas-
say following the manufacturer’s instructions (Human Procartaplex
customized panels; Invitrogen, Carlsbad, CA, USA). The plates were
read in a Luminex® 200 Instrument System using the xPONENT
software (Luminex Corporation, USA). The specific factors analyzed
were: Caspase-3, CD40L, EGF, FGF-2, FGF-23, G-CSF (CSF-3), GM-
CSF, GITRL, Granzyme B, GROα (KC/CXCL1), HGF, ICAM-1, IFN-γ , IL-
1α , IL-1β , IL-2, IL-6, IL-7, IL-8 (CXCL8), IL-10, MIP-1α (CCL3), MCP-
1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MIP-1β (CCL4), Osteopon-
tin (OPN), PDGF-BB, PECAM-1, P-Selectin, RANTES (CCL5), SDF-1α ,
Thrombopoietin (TPO), TGF-β , TNF-α , VCAM-1, VEGF-A and VEGF-
D.
For factor stability and consumption studies the specific panel
of factors analyzed were: EGF, G-CSF, GM-CSF, HGF, IFNg, MCP-1,
PDGF-BB, SDF-1a and VEGF-A.
2.8. Statistical analysis
When comparisons between all possible pairs of groups were
performed, the Tukey Honest Significant Differences (HSD) test was
used; while the Dunnett procedure was chosen when several com-
parisons against a control group were involved. Both statistical
methods control the family-wise error rate stemming from multi-
ple testing. Additionally, when single pairwise comparisons were
performed, a Studentś t-test was employed to compare means
136
Acta Biomaterialia 177 (2024) 132–147
amongst groups. Pearson’s rank correlation test was used to an-
alyze the correlation between the biological parameters. Unsuper-
vised agglomerative hierarchical clustering was performed to study
the similarities between the different samples and conditions using
the correlation distance and average linkage. The number of clus-
ters was estimated using the silhouette method and taking into ac-
count the biological information. Briefly, the silhouette coefficient
estimates the average distance between clusters, and plots them
over values of k ranging. Those with the highest coefficients rep-
resent the best clustering. For the representation of the cluster-
ing analysis, Principal Component Analysis (PCA), dendrograms and
heatmaps were plotted after scale-to-interval normalization. When
indicated, the confounding effect introduced by the inter-donor
variation was removed using a linear model to fit the data using
the removeBatchEffect function from the limma package [32]. Sta-
tistical analysis was performed with R version 4.2.1 (R Core Team,
2022) and Microsoft Excel. Figures were designed or partly cre-
ated using Servier Medical Art (provided by Servier, licensed un-
der a Creative Commons Attribution 3.0 unported license), Canva
(https://www.canva.com/es_es/), R and Adobe Illustrator CS4.
3. Results
3.1. The regenerative capacity of PRPs produced using a closed system
correlates to their molecular composition
Platelet-rich plasma (PRP) refers to many types of platelet-
based bioproducts, without making any distinction on their prepa-
ration and processing method, or their cellular and molecular com-
position, variables that surely influence their clinical outcome. In a
collaborative effort to offer a standardized production procedure,
we previously described the implementation of a closed-system
PRP preparation method using the infrastructure of our local blood
bank [30]. As part of the quality control of the process, we assessed
the concentration of some growth factors (GFs): EGF, HGF, PDGF-
BB, VEGF-A, VEGF-D and FGF-23, confirming at the molecular level
the acknowledged inter-donor variation of PRP-like products [30].
In order to assess the potential correlation of the molecular
composition of PRPs with their regenerative capacity, we first de-
cided to comprehensively characterize the PRPs (n=14) from dif-
ferent donors produced using the described method, extending the
list of GFs, cytokines, chemokines and immune modulators to 37
different molecules, which were measured using Multiplex Tech-
nology. Our results showed that PRP sample clustering was donor-
dependent, meaning that the molecular composition varied across
them, but stayed essentially identical among the aliquots from the
same donor (Fig. 2A). These data support not only the notion of
inter-donor variation, but also the previously described homogene-
ity in composition of the three aliquots prepared from the same
donor [30], which assures that a patient requiring a regime ther-
apy with serial applications is going to receive a PRP product of the
same quality. Principal Component Analysis (PCA) of the molecular
composition of PRPs further supports these results, as PRPs from
different individuals segregate, while aliquots from the same PRP
donor cluster together (Fig. S1A).
In order to evaluate whether the platelet count of PRPs may in-
fluence the inter-donor differences identified in factor composition,
we measured it on 10 PRPs from different donors (before the freez-
ing step to induce platelet lysis), and correlated it to the concentra-
tions of molecules measured with Multiplex Technology. Counter-
intuitively to what it could be anticipated, only the concentration
of 4 out of 37 molecules correlated significantly with platelet num-
ber, specifically EGF, VEGF-A, MIP-1β and Gro-α (Fig. 2B), while
for the other factors measured, no correlation was observed. These
results led us to hypothesize that, on the one hand, the PRPs ob-
tained with this method may have an optimal platelet yield en-
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al. Acta Biomaterialia 177 (2024) 132–147

Fig. 2. In-depth analysis of closed-system PRP. A. Heatmap depicting the hierarchical clustering of PRP samples after scale-to-interval normalization, based on their molecular
composition as measured by Multiplex Technology. The dendrogram on the top of the heatmap shows clustering of the samples based on their differential composition.
PRPs used in wound healing assays are indicated with asterisks at the bottom. B. Scatter plots representing the Pearson’s correlation between the platelet count and the
concentration (pg/mL) of EGF, Gro-α , VEGF-A and MIP-1α . C. Principal Component Analysis (PCA) of PRP samples used for wound healing assays based on their molecular
composition as measured by Multiplex Technology. D. Line graphs depicting the % of Scratch Covered Area (SCA) of wound healing assays at 12, 24, 36 and 48 hours. Control
and PRP-treated cultures, in the presence of 2 U/mL heparin, are shown. Experiments were carried out in standard culture conditions (supplemented with 10% FBS, left
graph) or in the absence of FBS (right graph). Data are presented as the mean + standard deviation (SD) of experimental replicates (n=3) from each culture condition, with
the exception of cultures treated with PRP 2 and PRP 5 (+ FBS), and PRP 6 (- FBS), where only the mean is represented (n=2). ∗ p<0.05; ∗ ∗ p<0.01; ∗ ∗ ∗ p<0.001. E. Graphs
representing the concentration of factors in PRP samples that induce low (PRP 1, 5 and 6) or high (PRP 2, 7, 10 and 12) proliferative responses compared to control cultures,
in the presence of FBS. These factors are the significant ones separating PRP 1 and PRP 5 from the rest in the PCA (Dim 2; B). Values measured in PRP 6 and PRP 10 are
indicated.

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A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al.
richment range, given that the donors are eligible for the blood
donation (i.e., there are not sensible differences in platelet count
between PRPs), and therefore, platelet-count related differences
would not be significant. Substantiating this notion, previous re-
sults in gonarthrosis patients receiving closed-system PRP, showed
that this standardized production method ensures a PRP product
with sufficient quality to produce positive clinical responses [33].
On the other hand, the presence of plasma proteins may limit
the detection of certain molecules making it difficult to observe a
sensitive correlation of the concentration of factors to the platelet
count as measured on individual PRPs.
Next, we performed in vitro wound healing/closure assays. We
tested available PRP samples with different molecular composi-
tion as evidenced by their hierarchical clustering (Fig. 2A, C and
Fig. S1A). We stimulated cultures of MRC-5 cells, in the presence
of FBS, with or without the different PRPs and the wound clo-
sure (gap area) was monitored. All PRPs enhanced cell prolifer-
ation compared to controls, however, their regenerative capacity
was markedly different amongst PRPs (Fig. 2D and Fig. S1B). PRPs
1, 5 and 6 seemed to exert a lower regenerative capacity compared
to PRPs 2, 7, 10 and 12. Furthermore, the regenerative capacity of
PRPs in the absence of FBS, i.e., as serum replacement (Fig. 2D),
maintained the same tendencies, although not so markedly, except
for PRP 1, who exerted the lowest regenerative response.
This supports the notion that the molecular composition of
PRPs conditions tissue regeneration, thus partly explaining the va-
riety in the clinical outcome of patients treated with these prod-
ucts. In an attempt to identify molecules that could be driving the
difference in regenerative response, we first looked into the fac-
tors of the PCA Component 2 (Dim2; Fig 2C), that separates PRP 1
and PRP 5 from the rest, which were MIP1-β , TGF-β and VCAM-1
(Fig. 2E). These PRPs showed additionally higher levels of factors
(i.e., inflammatory) in the larger factor cluster (see factor dendro-
gram of the heatmap, Fig. 2A), and were the ones that had less
regenerative potential, although with differences amongst cultures
when supplemented with FBS or not (Fig. 2D and Fig. S1B).
Of note, these wound healing assays were performed in the
presence of heparin (2 U/mL) to prevent the unavoidable clotting
of plasma factors, however, we observed that the addition of hep-
arin alone in control cultures was deleterious (Fig. S1C). In order to
circumvent this limitation, we grew cultures with PRP in the ab-
sence of heparin, and observed that the culture medium jellified,
creating a layer of a translucent fibrin jelly that still allowed imag-
ing with good resolution (Fig. S1D). For this reason, we decided to
continue without adding heparin to the cultures in further experi-
ments.
3.2. Regenerative capacity of PRP subfractions
It has been suggested that the plasma fraction of PRP-like prod-
ucts (i.e., plasma, PPP) accounts for most of the regenerative capac-
ity of the product, rather than the platelet-derived factors them-
selves [34]. Thus, our next goal was to compare the different sub-
fractions obtained with our standardized closed-system PRP pro-
duction method by characterizing them molecularly and by assess-
ing their regenerative capacity in vitro. For this comparison, we
included the original PRP and PPP. Next, an aliquot of PRP was
further processed by centrifugation. The pelleted platelets were
used to obtain plasma-free platelet lysate (PFP-L), and the super-
natant, tentatively named PRP Plasma to distinguish it from PPP, was
also included in the comparison (Fig. 1B). In Fig. 3A, we show
the hierarchical clustering of PRPs and derived subfractions based
on the concentration of measured molecules. A first observation
from this analysis is that there are evident differences between
PPP and PRP Plasma subfractions, which appears counterintuitive.
While it seems logical that the concentration of molecules should
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be equal amongst the two plasma subfractions, our results sug-
gest that PPP contains a low amount of factors in general, while
PRP Plasma (which is the subfraction containing the plasma ob-
tained from a third centrifugation performed on the PRP bioprod-
uct) contains a higher amount of some factors at levels compa-
rable to those measured in PRP (Fig. 3A, Fig. S2A and Table S1).
These results suggest that these factors that are in higher con-
centration in the PRP Plasma subfractions may be actively released
by platelets as a result of the additional processing procedure.
These factors include GFs (i.e., EGF, PDGF, VEGF-D or HGF) and
immunomodulators (interleukins and chemokines). On the other
hand, when comparing the PRP Plasma fraction with PRP and PFP-L
samples, the molecules that remained retained within the platelets
become evident (Fig. 3A). The comparison of the composition of
different subfractions allowed us to identify that ICAM-1/VCAM-1
and OPN/RANTES are mostly present in fractions containing plasma
(Table S1). In addition, the angiogenic factors VEGF-D and RANTES
are mainly present in PRP and PRP Plasma but not in PFP-L or PPP,
suggesting basal release into plasma by platelets (Fig. 3A-B). Strik-
ingly, PFP-L is enriched in GFs and other bioactive molecules in-
volved in tissue regeneration (Fig. 3A-B), compared to the original
PRP and other PRP-subfractions. However, the higher concentration
measured in PFP-L samples (prepared with the same number of
platelets), points toward additional reasons for the lower concen-
tration measured in PRP samples. Particularly, the concentration of
EGF was 7 times higher in PFP-L samples compared to PRP (683.8
pg/mL vs. 87 pg/mL respectively, see Fig. 3B and Table S1). Con-
sidering that PRP contains platelets in 100% plasma and PFP-L is
free of plasma (or may contain only traces), we propose that po-
tential plasma-protein sequestration of bioactive molecules might
explain the lower levels detected in PRP, thus interfering with the
detection in the Multiplex assay [35].
Next, we performed wound healing assays using the different
PRP subfractions as supplement. In assays performed in the pres-
ence of FBS, where we can assess the additive contribution of the
products tested to the serum-supplemented cultures, the regener-
ative potential of PRP was similar to that of the PRP Plasma frac-
tion, and in both cases slightly higher than the potential shown by
PPP, which is the poorest fraction in factor concentration (Fig. 3C-
D). These results are in concordance with the molecular profile of
the tested fractions. Of note, this difference is more acute in the
absence of FBS, i.e., when the different fractions are tested as re-
placement of the xenogeneic serum. As shown in Fig. 3D, the frac-
tions or products that exert the more favoring regeneration in vitro,
are those that contain platelet-derived factors, while PPP supports
cell-growth, however, below the growth supported by FBS alone.
At the functional level, the PFP-L achieved complete wound clo-
sure within 48 hours, showing the highest regeneration potential
and superiority compared to PRP (Fig. 3C-D) in the presence of
FBS. However, under starvation conditions (i.e., without FBS) the
regenerative potential of PFP-L was lower than that shown by the
plasma-containing subproducts, although still rescuing the absence
of FBS (Fig. 3D). We cannot assure whether this is due to the par-
ticular composition of the lysates, or to the absence of plasma
in the bioproduct, or serum in the culture conditions. Plasma (or
serum) may provide with components that may contribute in syn-
ergy with this bioproduct, as extrapolated from the cultures with
FBS, where it induces gap closure more efficiently.
3.3. Characterization of platelet secretomes (PLT-S): the platelet
signaling pathway activation drives their molecular composition and
regenerative capacity
The obtained results suggested that the fractions containing
platelet-derived factors, i.e., PRP, PRP Plasma and PFP-L, displayed
the highest regenerative capacity in vitro. Therefore, we next aimed
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al. Acta Biomaterialia 177 (2024) 132–147

Fig. 3. Molecular and functional analysis of PRP subfractions. A. Heatmap depicting the hierarchical clustering of the different PRP-subfractions based on their molecular
composition as measured by Multiplex Technology, after scale-to-interval normalization. The dendrogram on the top of the heatmap shows clustering of the samples based
on their differential composition. B. Comparison of growth factor (GF) enrichment in PRP and PFP-L bioproducts. The graphs represent the cumulative concentration (pg/mL)
of relevant groups of bioactive factors (based on molecular function) as measured in each platelet-based bioproduct. The average concentration of each factor is represented,
which is calculated from the concentrations measured in the subproducts (PRP and PFP-L) prepared from two independent donors (n=2). C. Representative images of the
wound healing assay and scratch closure monitoring over time in the presence of FBS. On the top of the panel, control culture images are shown at the beginning (T0) and
at the end (48 hours -hr-) of the assay. The scratch position is marked between dashed lines, and can be better seen on the T0 control image. Images of bioproduct-treated
cultures are shown below at the end of the assay (48 hr). D. Line graphs showing the % of Scratch Covered Area (SCA) of wound healing assays at 12, 24, 36 and 48 hours of
control and bioproduct-stimulated cultures. Experiments were carried out in standard culture conditions (supplemented with 10% FBS, left graph) or in the absence of FBS
(right graph). Data are presented as the mean + standard error of the mean (SEM) of cultures treated with PRP subfractions obtained from different donors (n=4), with the
exception of control cultures and cultures treated with PFP-L (- FBS), where n=3. ∗ p<0.05; ∗ ∗ p<0.01; ∗ ∗ ∗ p<0.001.

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at producing a platelet-derived bioproduct, free of plasma and
undesirable platelet components, made of an enriched platelet-
granule secretome generated by activating platelets to induce their
degranulation (PLT-S). As a quality control of appropriate cargo de-
livery in our PLT-S, we evaluated platelet degranulation by flow cy-
tometry. For this purpose, surface expression of CD62P and CD63
was assessed upon platelet stimulation, as an indirect measure of
granule release (alpha granules -CD62P-, and dense granules and
lysosomes -CD63-); (Fig. S2B). PLT-S samples were selected for fur-
ther molecular characterization by Multiplex Technology and func-
tional studies when unstimulated platelets did not show any signs
of pre-activation, but displayed normal degranulation upon stimu-
lation.
PLT-S samples were prepared in accordance with our produc-
tion method, using platelets from whole blood (WB) as start-
ing material and stimulating them with several receptor agonists.
Specifically, we stimulated GPVI with convulxin (CVX) and high-
dose collagen (Coll-High), integrin α 2β 1 with low-dose collagen
(Coll-Low), PAR1 with TRAP6, CLEC2 with aggretin A (AggA), in-
tegrin α IIbβ 3 with PMA, and the von Willebrand factor receptor
(vWF-R; GPIB-V-IX) with ristocetin; we also included unstimulated
platelets as control (Fig. S2B-C). Subsequently, we studied the com-
position of the PLT-Ss obtained in response to different stimuli. The
molecular characterization of samples showed that FGF-2 and IL-
8 were either not present, or below the detection limit (see Ta-
ble S1), hence these factors were filtered out for further analysis.
Of note, although we observed a clear inter-donor variability (Fig.
S3A), the data also shows a clustering that depends on the platelet
response or status: active aggregation/degranulation or agglutina-
tion/resting platelets, with the exception of one sample stimulated
with ristocetin that clustered with the aggregation/degranulation
samples (Fig. S3B). Once the inter-donor variability was removed
from the data, the multiplex quantification results showed a differ-
ent degranulation profile depending on the platelet agonist used,
which is visualized by hierarchical clustering in the heatmap and
PCA plots (Fig. 4A-B). It should be noted that the PCA generated
with the average of the raw data (pg/mL) resulted in the same
sample clustering, which confers reliability to the batch effect re-
moval analysis (Fig. S3C). Specifically, our results showed that the
stimulation with CVX and collagen (high and low doses) generated
a similar secretion profile, result that is consistent with the cross-
talk between the signal transduction pathways activated by those
agonists. Furthermore, the stimulation with TRAP-6 and AggA gen-
erated a secretome with similar composition pattern, characterized
by an enrichment in the adhesion proteins ICAM-1/VCAM-1. Fi-
nally, the stimulation with PMA generated a unique secretion pro-
file characterized by lower levels in some growth factors (i.e., EGF,
VEGF-D and PDGF-BB, among others). In contrast, when platelets
were stimulated with ristocetin, which activates the vWF-R com-
plex, the concentration of most of the bioactive factors drastically
decreased to levels comparable to those measured in unstimulated
platelets (Table S1 and Fig. S3D). The response to ristocetin was not
unexpected, as activation of vWF-R by this agonist does not trigger
a secretion response per se, but rather it induces platelet aggluti-
nation [36]. As evidenced in Fig. S2B, the stimulation with risto-
cetin resulted in the creation of an agglutinate-mesh where, as we
hypothesized, any released bioactive factors would be sequestered.
Thus, the physical characteristics of aggregate formation might also
influence factor release.
We next evaluated the in vitro regenerative potential of the PLT-
Ss and whether the differences observed in composition would
promote associated grades of regeneration. We performed the as-
says in the presence of FBS at first, because we intended to see dif-
ferences in their potentiation capacity rather than evaluating their
serum replacement capacity. Our results show that the wound clo-
sure dynamic was highly dependent on the composition of the bio-
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Acta Biomaterialia 177 (2024) 132–147
product, which in turn associated to the agonist used to activate
the platelet cargo degranulation (Fig. 4C and Fig. S3E). As we ex-
pected, based on the quantification of bioactive factors by Multi-
plex Technology, the secretome obtained through the stimulation
of GPVI by CVX resulted in the most mitogenic response, reaching
a 96% of wound closure compared to the 66% of the control. Sim-
ilar results were obtained with collagen. Likewise, the secretome
obtained with PMA showed a high regenerative potential, with a
wound closure dynamic similar to that of the PLT-S obtained with
AggA. Strikingly, the secretome obtained with TRAP6 resulted in
the lowest regenerative potential (maximal scratch covered area
85%). Overall, our results highlight the fact that the regenerative
capacity of PLT-Ss differs depending on the agonist used to induce
platelet degranulation, because that determines its composition. Fi-
nally, and in concordance with the poorer composition, the super-
natant obtained after platelet stimulation with ristocetin showed a
wound closure dynamic similar to control cells (Fig. S3E-F).
3.4. The platelet source used to prepare PLT-S conditions its
composition and in vitro regenerative capacity
We next studied whether the source of platelets used to pre-
pare PLT-Ss would condition the quality and regenerative capac-
ity of PLT-Ss themselves. With this objective, we prepared PLT-
Ss from two different sources, whole blood (PLT-SWB ), or closed-
system PRPs before freezing (PLT-SPRP ); and using CVX, PMA and
TRAP6 as stimuli. Molecular characterization of the bioproducts
by Multiplex Technology was performed. As previously observed,
FGF-2 and IL-8 were not detected in PLT-S samples (see Table S1),
hence these factors were filtered out for further analysis. As shown
in Fig. 5A, the hierarchical clustering of samples based on their
molecular composition shows that the platelet source is a vari-
able that significantly influences the composition of the final bio-
product. Specifically, the PLT-SWB showed a higher concentration of
most GFs and bioactive molecules, which translates into a greater
regenerative potential (in the presence of FBS) (Fig. 5B). This sug-
gested that the PRP manufacture procedure caused basal platelet
degranulation that occurred either during the 24 hour storage from
collection to processing, or during the production itself. Since we
discarded the soluble fraction to prepare PLT-SPRP , most probably
we lost the released factors in our final product. Corroborating our
previous results, the factors were represented in the PLT-Ss in dif-
ferent proportions, not only due to the influence of the platelet
source, but also due to the signaling pathway activation (i.e., the
stimulus used to produce the secretome, Fig. 5C).
3.5. In vitro stability and consumption of factors from PLT-S
bioproducts
In an attempt to determine the stability and consumption of
factors from PLT-Ss in culture, we tested different PLT-Ss as de-
scribed in the Materials and Methods section, and we selected a
panel of GFs to monitor using Multiplex Technology. As shown
in Fig. S4A, MRC-5 cells themselves produced some of the factors
contained in our panel, and thus, present in PLT-Ss, i.e., HGF, MCP-
1, SDF-1α and VEGF-A, which may preclude the analysis of factors
included in our panel. As expected, the concentrations measured in
supernatants were increased in cultures with a higher number of
cells. Next, we set out to evaluate the stability of factors in cultures
devoid of cells. We observed high variability in the dynamics of the
concentration of factors that we were able to measure in cultured
medium supplemented with the tested PLT-Ss after 48 hours, i.e.,
EGF, PDGF-BB and VEGF-A (the dilution factor still allowed their
detection in supernatants). From the results obtained, it appeared
that the stability or decline of a single factor is not associated with
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al. Acta Biomaterialia 177 (2024) 132–147

Fig. 4. Composition and functional analysis of platelet secretomes from whole blood (PLT-SWB ). A. The heatmap depicts the hierarchical clustering of PLT-SWB samples based
on their molecular composition as measured by Multiplex Technology, after batch-effect removal and scale-to-interval normalization. The dendrogram on the top of the
heatmap shows clustering of the samples based on their differential composition. Samples from each donor (1 to 4) are indicated. The agonist used to produce secretomes
in each case, is also indicated: A, Aggretin A; L, Collagen Low concentration; H, Collagen High concentration; X, Convulxin; T, TRAP6; P, PMA. B. Principal component analysis
(PCA) of PLT-SWB samples after batch-effect-removal and scale-to-interval normalization. Component 2 (Dim 2) separates most secretomes obtained with Collagen High
and Low concentration (Coll-High and Coll-Low) and Convulxin (top quadrant), from those obtained with TRAP6, Aggretin A or PMA (bottom quadrant). C. Representative
microscopic images of control wound healing assays at the beginning (T0) and at the end (48 hr) of cultures, in the presence of FBS. The scratch position is marked between
dashed lines and can be better visualized on the T0 control image. Representative images of cultures treated with PLT-Ss obtained with different agonists are shown at the
end of the assay (48 hr). The line graphs show the % of Scratch Covered Area (SCA) of wound healing assays at 12, 24, 36 and 48 hours of control and PLT-S treated groups.
Data are presented as the mean + standard deviation (SD) of experimental replicates (n=3). ∗ p<0.05; ∗ ∗ p<0.01; ∗ ∗ ∗ p<0.001.

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Fig. 5. The platelet source is a key driver in the composition of PLT-Ss. A. Heatmap depicting the hierarchical clustering of PLT-SWB and PLT-SPRP samples based on their
molecular composition as measured by Multiplex Technology, after scale-to-interval normalization. Samples from each donor (1 to 4 and 15 to 18) are indicated. The agonist
used to produce secretomes in each case, is also indicated: X, Convulxin; T, TRAP6; P, PMA. B. Line graphs showing the % of Scratch Covered Area (SCA) of wound healing
assays at 12, 24, 36 and 48 hours cultured with PLT-SWB or PLT-SPRP -obtained using CVX as agonist- (in the presence of FBS). The upper graph shows the SCA from cultures
supplemented with PLT-SWB or PLT-SPRP and respective controls, and the lower graph shows the normalized SCAs after setting respective controls to 0 at each time point.
Data are presented as the mean + standard deviation (SD) of control cultures and cultures treated with bioproducts obtained from different donors (n=3). ∗ p<0.05; ∗ ∗
p<0.01. C. Lollipop plots illustrating the composition variation (Log2FC WB/PRP ratio) of PLT-Ss depending on the platelet source and conditioned by the platelet-receptor
agonist used for stimulation.

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its initial concentration, which suggests that the global composi-
tion of the bioproduct conditions the stability of single factors (Fig.
S4B). Finally, to evaluate factor consumption we set cultures with
a low (as in our wound healing assays) or high number of MRC-
5 cells and supplemented them with the tested PLT-Ss. The results
obtained show that factors were not necessarily exhausted after 48
hours in the cultures with a lower cell number (i.e., wound heal-
ing assay condition). When they were exhausted, it was not related
undoubtedly to a lower initial concentration of the given factor.
In cultures with a higher cell number, the reduction in concen-
tration of factors was noticeable, but it not always led to exhaus-
tion. Finally, in the case of VEGF-A, and not always, the addition
of PLT-S might induce the production of the factor itself by MRC-5
cells, thus making it impossible to discern the remaining VEGF-
A from the PLT-S supplement or the VEGF-A newly produced by
MRC-5 cells (Fig. S4C). Overall, this preliminary data suggests that
the global composition of the bioproduct may condition the stabil-
ity and consumption of single factors, which will also be cell-type-
or tissue-dependent (Fig. S4).
3.6. Regenerative capacity and molecular composition across the
different platelet-based subproducts
Next, we compared PLT-Ss with our gold standard reference,
closed-system PRP, as well as the other relevant PRP subfractions,
to better assess the regenerative capacity of platelet-based bio-
products in a comprehensive and comparative manner. To this
end, we prepared additional formulations in which the number
of platelets was even between the different bioproducts. We en-
gineered, from the same donor source, PRP and PFP-L contain-
ing the same concentration of platelets as we used to prepare
PLT-S (Custom PRP and Custom PFP-L, respectively) and, on the other
hand, we prepared PLT-S containing the same concentration of
platelets in PRP, which is on average 3X higher (namely Supra PLT-
SPRP ; see Fig. 1C-D). The comparison of the molecular profile of
bioproducts showed that PRP samples (PRP and Custom PRP) and
PFP-L samples (PFP-L and Custom PFP-L) clustered together respec-
tively, although the factor concentration was lower in the respec-
tive custom products, i.e., prepared with an original lower number
of platelets (Fig. 6A). As we observed before, the PLT-SWB showed
the most enriched composition in GFs, and if we consider the cu-
mulative concentration of the pool of GFs analyzed, this variable
would be similar to that obtained in Supra PLT-SPRP and PFP-L, where
the number of initial platelets is approximately 3X higher than in
PLT-SWB (Fig. 6B). However, it is important to acknowledge that
the proportion of the individual GFs in each sample was different.
While PLT-SWB showed a higher concentration of PDGF-BB, G-CSF
and TGF-β , Supra PLT-SPRP displayed a higher concentration of VEGF-
A. Instead, PFP-L contained a higher concentration of TPO and EGF
(Fig. 6B and Table S1). Of note, all subfractions were prepared from
the same donors, except the PLT-SWB , so we cannot rule out donor-
dependent variation contributing to the composition differences.
Comparison between bioproducts prepared with the same initial
platelet concentration, corroborated that cytokines and chemokines
were also detected in greater proportion in secretome samples (Fig.
S5A-B). It is interesting to highlight that PLT-SWB contained a lower
proportion of the proinflammatory cytokine IL-6 and increased lev-
els of GITRL and SDF-1α , key factors involved in cell migration and
proliferation [37,38].
Based on this molecular characterization, we hypothesized that
this platelet-based bio-product, PLT-S, could have a higher regen-
erative potential compared to PRP, as it is exclusively enriched in
platelet granule molecules. To evaluate this hypothesis, we set out
to compare the functional profile of the different platelet-based
bioproducts in in vitro wound healing assays. Our results showed
that the wound closure dynamic in cultures supplemented with
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Acta Biomaterialia 177 (2024) 132–147
each subproduct, in synergy with FBS, was completely in line with
the level of GFs measured in each of them (Fig. 6C and S5D).
Specifically, PLT-SWB and PFP-L outpassed the PRP-related bioprod-
ucts. In concordance with the cumulative concentration of mea-
sured GFs, cytokines and chemokines, the regenerative capacity of
Custom PRP and PLT-SPRP was inferior compared to PRP, PFP-L and
PLT-SWB . Surprisingly, we observed that Custom PFP-L exerted an in-
hibitory effect on cell proliferation compared to the control assays.
Of note, in this particular bioproduct, the concentration of most
factors was lower than in any other subproduct, while the concen-
tration of Caspase-3 remained at the levels of the ones measured
in related PFP-L fractions (Fig. S5C).
Next, the regenerative capacity was assessed in the absence
of FBS, with those subproducts that showed similar or superior
regeneration potential to PRP in the previous analysis. We com-
pared PRP, PFP-L, PLT-SWB and Custom PRP (Fig. 6D and Fig. S5E).
Our first observation was that all bio-products were able to sup-
port cell proliferation in the absence of FBS, proving their poten-
tial use as xenogeneic serum replacement (Fig. S5E). However, the
proliferation curve showed different kinetics amongst subproducts.
Importantly, and additional to Fig. 3D, where it was shown that
PFP-L regenerative capacity was outpassed by PRP subproducts,
we observed in this comparative analysis that the regenerative ca-
pacity correlated with platelet count on customized products, i.e.
Custom PRP displayed a slightly lower regenerative capacity than PRP
(Fig. S5E). Interestingly, the potential to stimulate cell proliferation
and/or migration shown by PLT-SWB was higher than that shown
by the rest of PRP-based bioproducts (Fig. 6D and Fig. S5E). The su-
periority of PLT-S (specially the one obtained from WB) compared
to not only PRP, but also to PFP-L, in terms of regenerative capacity,
is remarkable, as PFP-L is prepared with an initial higher number
of platelets.
4. Discussion
The use and demand of platelet-based bioproducts in regen-
erative medicine has increased in recent years. However, the use
of PRP remains highly controversial due to a lack of standard-
ization of preparation and application methods, which results in
highly variable PRP products (in terms of purity, content, quality
and safety), which severely impact their potential clinical efficacy
[24,26]. Furthermore, as of today, it is very difficult to establish the
real clinical benefits of these therapies, as the lack of characteriza-
tion of the bioproducts and a detailed description of the produc-
tion methods is a constant in the literature, precluding cross-study
comparisons. To avoid that skepticism towards platelet-based bio-
products grows along with their increasingly popular use in regen-
erative medicine, there is an urgent need for a better characteri-
zation of the products used in each study and the need of stan-
dardized production methods. In this study, we characterize the
in vitro regenerative capacity of platelet-based bioproducts and re-
lated subfractions, and associate it with their molecular composi-
tion profile.
As a starting point, we first characterized PRP produced in a
closed system with a standardized procedure, which relays on the
equipment of a certified blood bank (closed-system PRP, WB PRPFT2
according to the nomenclature proposed by us [24,30]). Our re-
sults support the notion of inter-donor variation at the molecu-
lar and functional level, while they also highlight the fact that a
standardized method to produce PRP assures a certain quality of
the product, minimizing variation due to other variables, such as
the platelet count in the original platelet-based bioproduct. In this
sense, some studies have suggested an impact of age or gender
on PRP GF levels. Evanson et al. reported that the concentration of
certain GFs was higher in women compared to men (at the same
platelet ratio of PRP vs whole blood for both genders) as well as
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al. Acta Biomaterialia 177 (2024) 132–147

Fig. 6. Molecular profile and regenerative capacity across different platelet-based subproducts. A. Heatmap depicting the hierarchical clustering after scale-to-interval nor-
malization of the different subproducts based on their molecular composition as measured by Multiplex Technology: PRP-based (PRP and Custom PRP), PFP-L formulations
(PFP-L and Custom PFP-L) and PLT-Ss (PLT-SWB , PLT-SPRP and Supra PLT-SPRP ). The dendrogram on the top of the heatmap shows clustering of the samples (mean values) based on
their differential composition. The agonist used to produce secretomes in each case, is indicated: A, Aggretin A; H, Collagen High concentration; L, Collagen Low concentra-
tion; X, Convulxin; T, TRAP6; P, PMA. B. The bar graph represents the cumulative concentration (pg/mL) of GFs detected by Luminex in the different platelet bioproducts.
The average concentration of each factor is represented, which is calculated from the concentrations measured in the subproducts prepared from four independent donors
(n=4), with the exception of PFP-L and Custom PFP-L (n=2). C. Line graphs shows the % of Scratch Covered Area (SCA) of wound healing assays at 12, 24, 36 and 48 hours of
control and treated groups in addition to 10% FBS, as standard culture conditions. The graph on the top compares the SCA of cultures supplemented with PLT-Ss -obtained
from whole blood (WB) or PRP sources using CVX as agonist- with those supplemented with Custom PRP or Custom PFP-L (i.e., obtained from the same number of platelets as the
PLT-Ss) and respective controls. The graph on the bottom shows the results from the same assays, after normalization to respective controls, including cultures supplemented
with regular PRP and PFP-L. Data are presented as the mean + standard error of the mean (SEM) of biological replicates (n=4, except for PLT-Ss where n=3). ∗ p<0.05; ∗ ∗
p<0.01; ∗ ∗ ∗ p<0.001. D. Representative microscopic images of wound healing assays in MRC-5 fibroblast cell cultures at the beginning and after 24 and 48 hours, and in the
absence of FBS supplementation. Untreated cells were taken as control. The line graphs show the % of SCA of wound healing assays at 12, 24, 36 and 48 hours of control
and treated groups. Data are presented as the mean + standard error of the mean (SEM) of biological replicates (n=4, except for PLT-SWB where n=3). Statistical analysis
was applied to compare PLT-SWB with Custom PRP; ∗ p<0.05; ∗ ∗ p<0.01.

144
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al.
in younger people (≤ 25 years old) [39]. Additionally, other stud-
ies reported a negative correlation between age and PDGF-BB and
IGF-1 levels [40,41]. A limitation of our study is that we could not
possibly assess the impact of age and gender, as we used inter-
rupted donations or discard material from the blood bank, without
inclusion requirements. Another aspect to take into consideration
is that our study focused on the analysis of 37 growth factors and
cytokines, which precludes data on other components (other pro-
teins, RNA, miRNA and metabolites) that may influence the regen-
erative capacity of each product [42–45].
Our next objective was to assess the regenerative capacity
of the different subfractions obtained from PRPs produced in a
closed-system. With that aim, fresh-PRP samples were dissected
into their components, plasma and platelets, to identify the contri-
bution of each fraction to the wound healing process. Importantly,
our experimental design allowed the comparison of different frac-
tions obtained from the same donor. Our study showed that PPP
worked itself as a potent stimulator of cell proliferation. This ob-
servation is in line with previous studies showing the effective-
ness of PPP in the healing process [46–48]. Indeed, although PPP
by definition contains a very low concentration of platelets and,
therefore, smaller quantities of GFs, it still represents a reservoir
of bioactive molecules (such as PDGF-BB, VEGF-A, TGF-β ), which
may be responsible for the observed effect [49]. In addition, the
plasma compartment contains high levels of fibrinogen; interest-
ingly, the fibrin network that it forms upon thrombin stimulation,
has been postulated as a controlled release system beneficial for
correct wound healing [50,51].
Of relevance, our results also explain the observations described
in other studies that claim that the plasma fraction contributes to
the major regenerative potential capacity of these products. In fact,
basal degranulation of platelets due to processing, and release of
these factors to the so-called “plasma” fraction, might be respon-
sible for the described contribution of “plasma” to the regenera-
tive process on those studies. Our experimental approach revealed
that the additional sample processing to separate these fractions
resulted in a PRP-like bioproduct (PRP Plasma in our experimental
approach), containing additional platelet-derived cargo molecules
and displaying higher regenerative capacity compared to PPP, and
similar to that of the reference PRP. This result is of special rele-
vance, given that there is not sufficient research studying the rel-
ative contribution of both plasma and platelets to the regenera-
tive potential of PRP, and many studies obtain plasma from platelet
concentrates, downplaying the role of platelets in the observed ef-
fects [34,52]. The blood plasma fraction shall always contain solu-
ble platelet-derived factors, naturally, and it should be considered
that the processing of samples to separate platelets from plasma
might induce additional platelet activation and degranulation into
the plasma fraction. It is thus very difficult to separate cleanly
a plasma fraction free of platelet-secreted molecules, and caution
should be taken when interpreting results from these studies.
This dissection also showed that the platelet lysate fraction,
PFP-L, induced a lower regenerative capacity, and even appeared
to be deleterious to the cultures. Of note, we identified in this par-
ticular bioproduct a higher concentration of Caspase-3, which has
been recently shown to exert non-apoptotic functions related to
the proliferation and modulation of cell growth [53]. In addition,
it has been previously described that an enhanced expression of
Caspase-3 in hypertrophic scars and keloid induces apoptosis of fi-
broblasts [54]. Taken together, and in agreement with other stud-
ies, our data support a personalized approach to PRP therapy and
underscore the need for a greater understanding of the relation-
ships between PRP composition and clinical outcomes.
The autologous use of PRP bioproducts, as required by local reg-
ulatory establishments, may also impede its prescription in certain
patients. Certain pathological conditions might influence platelet
145
Acta Biomaterialia 177 (2024) 132–147
quality and reactivity, and the use of autologous products might
not always be the most suitable therapeutic source [27,28,55]. Con-
sequently, there is an increased clinical interest in the development
of platelet-based bioproducts that could be used universally (allo-
geneic), and would be more specific or targeted to the pathology
being treated, in an effort to improve and personalize the therapy.
Interestingly, the dissection of PRP subfractions allowed us to
identify the fractions that actively contribute to tissue regenera-
tion, i.e., those that contain undoubtedly platelet-derived factors.
Therefore, we developed a method to generate plasma-free se-
cretomes (PLT-S; PCT/IB2022/057936). Contrarily to methods de-
scribed by other authors, we did not use apyrase or prostaglandin
E2 (PGE2) to minimize platelet activation during the manipula-
tion steps (pipetting and centrifugation) [14]. We consider that
the use of these compounds may result in slightly anergic platelet
responses upon stimulation. From our point of view, optimized
washing protocols with minimum steps and the use of an appro-
priate buffer (HEPES-Glucose or HEPES-Tyrode’s buffer) prevents
the pre-activation and destruction of platelets, as well as con-
tamination by erythrocytes and leukocytes [24,56]. Another step
avoided in our protocol, and a matter of concern from our perspec-
tive, is the interruption of the platelet stimulation with ice [21,57].
It is widely known that cold temperatures promote platelet activa-
tion and aggregation [58,59]. Thus, from our point of view, placing
reactions on ice would introduce an additional stimulation step,
which could affect the composition and the subsequent regener-
ative capacity of the bioproduct. Furthermore, we would not be
able to separate the ice-activating interference and its contribution
to the agonist-induced degranulation. Our analysis revealed a dif-
ferent platelet secretion profile and functional response of PLT-Ss
in an agonist-dependent manner. This result is consistent with the
observations made by other authors [21].
Another key aspect in the preparation of platelet-based bio-
products is the choice of the platelet source. As a consequence
of a spontaneous release, as we hypothesize, platelets from fresh
closed-system PRP have a lower degranulation capacity in response
to agonists. Thus, PLT-Ss prepared from PRP samples have lower
concentration of factors measured, as we showed, compared to
PLT-Ss produced directly from whole blood. This observation is of
relevance, since PRP and other platelet derived products are be-
ing considered to be preparedfrom platelet concentrates that are
expired for transfusion. While this option would assure a circular
economy of the process, it should be acknowledged that the obten-
tion method should be adapted specifically to the platelet source,
in order to obtain a product of certain quality, i.e., pooling concen-
trates or donations for large-scale production of off-the-shelf prod-
ucts with certified quality. This also applies to the variation in sta-
bility and consumption of factors as observed in vitro, which ap-
pear to be dependent on the bioproduct composition, and hence,
dependent on inter-donor variation.
Lastly, we compared the composition profile and functionality
of PRP, PFP-L and PLT-S subproducts. In general, GF levels were
higher (approximately 3-fold) in PLT-SWB samples compared to
Custom PRP (prepared with the same number of platelets). We hy-
pothesized that the low levels of GFs and other immune media-
tors detected in PRP might be due to the presence of the plasma
fraction. Several studies report that the association of GFs with
specific plasma-binding proteins, blocks the interaction between
the GF and its cell-surface receptor and modulates their functional
activity [35,60]. Interestingly, we observed a superiority of PLT-S
compared to PRP in vitro, with some remarks. Thus, the depletion
of plasma in PLT-S constitutes a great advantage compared to PRP
in terms of bioavailability and functionality of GFs. Nevertheless, it
seems pertinent to highlight that, in the PRP, the growth factors
sequestered by plasma proteins might be gradually released and
protected against degradation for a longer time, allowing longer
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al.
storage. In this sense, we envision that the encapsulation of PLT-
S in nanoparticles made of different biomaterials (i.e., liposomes,
synthetic platelets, etc.) would improve their absorption, stability
and functionality, while allowing to treat non-accessible tissues
or locations, in a targeted and personalized manner. Other post-
production procedures (i.e., lyophilization), would allow the pro-
duction of PLT-S that could be stored at the clinical practice in
order to be available for immediate use. Alloimmunization is an-
other important issue, which is reduced to almost zero risk when
using plasma-free bioproducts, and this characteristic would allow
the allogeneic use of PLT-S.
Overall, our findings support the idea of using PLT-S for thera-
peutic applications as an alternative to the current autologous ap-
plication of PRP. Given its characteristics, PLT-S represents a plau-
sive option to explore novel applications in regenerative medicine
and other clinical fields: it would allow allogeneic use and the pro-
duction method could be optimized to minimize batch-to-batch
variation, or to incorporate post-processing steps to allow nanoen-
capsulation (targeted delivery) or storage with off-the-shelf pur-
poses. Future studies in more complex cellular and tissue settings
(3D reconstructions, organoids) and/or in vivo models are needed
to fully understand the regenerative capacity of this platelet-
based bioproduct, and the specific production and post-production
methodology adaptations for long-term storage and each singular
clinical application.
Author contributions
Conceptualization, L.G. and A.A.-H; investigation and method-
ology, A.A.-H. and L.G.; resources and reagents, J.F.-F., A.M.O.-P.,
M.C.M.-T., A.L.-V. and J.A.E.; performance of experiments, A.A.H:
and G.C.-A.; formal analysis, A.A.-H., P.M.-B. and L.G.; writing—
original draft preparation, A.A.-H. and L.G.; writing—review and
editing, A.A.-H., P.M.-B and L.G.; supervision, L.G. All authors have
read and agreed to the published version of the manuscript.
Declaration of competing interest
The authors declare the following financial interests/personal
relationships which may be considered as potential competing in-
terests: A.A.-H. and L.G. are co-founders of the recently constituted
spin off company, Platelet Biotechnologies S.L. (PlaBiTe). The rest of
authors have no competing interest to disclose.
Data availability
The raw data required to reproduce these findings are available
to download from Table S1.
Funding
This study was partially supported by an I+D Excelencia 2020
project grant (PID2020- 117265GB-I00, Ministerio de Ciencia e In-
novación -Spain-) to L.G. A.A.-H. was supported by said grant.
P.M.-B. is supported by a Severo Ochoa Grant (Consejería de Cien-
cia, Innovación y Universidad del Principado de Asturias, PA-20-
PF-BP19-014). J.A.E. is financially supported by the German Re-
search Foundation (DFG – Project-ID 194468054 – SFB 1009project
A09) and by the Interdisciplinary Center of Clinical Research (IZKF
grant: Ebl-A/009/21). Patent application fees (PCT/IB2022/057936
and P202130806) were partially covered by Technology Trans-
fer grants (Consejería de Ciencia, Innovación y Universidad del
Principado de Asturias AYUD/2021/57101, AYUD/2022/26253 and
AYUD/2022/33562).
146
Acta Biomaterialia 177 (2024) 132–147
Acknowledgements
The authors thank all the institutions and personnel involved
in this study, with special emphasis to the Centro Comunitario de
Sangre y Tejidos de Asturias (Oviedo, Spain) and Immunology Ser-
vice, Hospital Universitario Central de Asturias (Oviedo, Spain), for
their assistance with the Luminex® Analyzer.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.actbio.2024.01.029.
References
[1] P.E.J. van der Meijden, J.W.M. Heemskerk, Platelet biology and functions: new
concepts and clinical perspectives, Nat. Rev. Cardiol. 16 (3) (2019) 166–179.
[2] M. Leslie, Cell biology. beyond clotting: the powers of platelets, Science 328
(5978) (2010) 562–564.
[3] K. Suzuki-Inoue, N. Tsukiji, T. Shirai, M. Osada, O. Inoue, Y. Ozaki, Platelet
CLEC-2: roles beyond hemostasis, Semin. Thromb. Hemost. 44 (2) (2018)
126–134.
[4] J.W. Semple, J.E. Italiano Jr., J. Freedman, Platelets and the immune continuum,
Nat. Rev. Immunol. 11 (4) (2011) 264–274.
[5] L.J. Gay, B. Felding-Habermann, Contribution of platelets to tumour metastasis,
Nat. Rev. Cancer 11 (2) (2011) 123–134.
[6] C.N. Jenne, R. Urrutia, P. Kubes, Platelets: bridging hemostasis, inflammation,
and immunity, Int. J. Lab. Hematol. 35 (3) (2013) 254–261.
[7] L.F. Brass, H. Jiang, J. Wu, T.J. Stalker, L. Zhu, Contact-dependent signaling
events that promote thrombus formation, Blood Cells Mol. Dis. 36 (2) (2006)
157–161.
[8] S.P. Jackson, W.S. Nesbitt, S. Kulkarni, Signaling events underlying thrombus
formation, J. Thromb. Haemost. 1 (7) (2003) 1602–1612.
[9] E.M. Battinelli, B.A. Markens, J.E. Italiano Jr., Release of angiogenesis regulatory
proteins from platelet alpha granules: modulation of physiologic and patho-
logic angiogenesis, Blood 118 (5) (2011) 1359–1369.
[10] J.A. Coppinger, P.B. Maguire, Insights into the platelet releasate, Curr. Pharm.
Des. 13 (26) (2007) 2640–2646.
[11] A. Sharda, R. Flaumenhaft, The life cycle of platelet granules, F10 0 0Res. 7
(2018) 236.
[12] D.M. Maynard, H.F. Heijnen, M.K. Horne, J.G. White, W.A. Gahl, Proteomic anal-
ysis of platelet alpha-granules using mass spectrometry, J. Thromb. Haemost.
5 (9) (2007) 1945–1955.
[13] D.M. Maynard, H.F. Heijnen, W.A. Gahl, M. Gunay-Aygun, The alpha-granule
proteome: novel proteins in normal and ghost granules in gray platelet syn-
drome, J. Thromb. Haemost. 8 (8) (2010) 1786–1796.
[14] O. Pagel, E. Walter, K. Jurk, R.P. Zahedi, Taking the stock of granule cargo:
platelet releasate proteomics, Platelets 28 (2) (2017) 119–128.
[15] E.M. Battinelli, J.N. Thon, R. Okazaki, C.G. Peters, P. Vijey, A.R. Wilkie, L.J. Noet-
zli, R. Flaumenhaft, J.E. Italiano, Megakaryocytes package contents into sepa-
rate alpha-granules that are differentially distributed in platelets, Blood Adv. 3
(20) (2019) 3092–3098.
[16] P. Handagama, R.M. Scarborough, M.A. Shuman, D.F. Bainton, Endocytosis of
fibrinogen into megakaryocyte and platelet alpha-granules is mediated by al-
pha IIb beta 3 (glycoprotein IIb-IIIa), Blood 82 (1) (1993) 135–138.
[17] P. Handagama, D.F. Bainton, Y. Jacques, M.T. Conn, R.A. Lazarus, M.A. Shuman,
Kistrin, an integrin antagonist, blocks endocytosis of fibrinogen into guinea
pig megakaryocyte and platelet alpha-granules, J. Clin. Invest. 91 (1) (1993)
193–200.
[18] B.A. Bouchard, N.T. Meisler, M.E. Nesheim, C.X. Liu, D.K. Strickland, P.B. Tracy,
A unique function for LRP-1: a component of a two-receptor system mediating
specific endocytosis of plasma-derived factor V by megakaryocytes, J. Thromb.
Haemost. 6 (4) (2008) 638–644.
[19] G.L. Klement, T.T. Yip, F. Cassiola, L. Kikuchi, D. Cervi, V. Podust, J.E. Ital-
iano, E. Wheatley, A. Abou-Slaybi, E. Bender, N. Almog, M.W. Kieran, J. Folk-
man, Platelets actively sequester angiogenesis regulators, Blood 113 (12) (2009)
2835–2842.
[20] H. Heijnen, P. van der Sluijs, Platelet secretory behaviour: as diverse as the
granules ... or not? J. Thromb. Haemost. 13 (12) (2015) 2141–2151.
[21] D. Jonnalagadda, L.T. Izu, S.W. Whiteheart, Platelet secretion is kinetically
heterogeneous in an agonist-responsive manner, Blood 120 (26) (2012)
5209–5216.
[22] R. Alves, R. Grimalt, A review of platelet-rich plasma: history, biology, mecha-
nism of action, and classification, Skin. Appendage Disord. 4 (1) (2018) 18–24.
[23] J. Alsousou, A. Ali, K. Willett, P. Harrison, The role of platelet-rich plasma in
tissue regeneration, Platelets 24 (3) (2013) 173–182.
[24] A. Acebes-Huerta, T. Arias-Fernandez, A. Bernardo, M.C. Munoz-Turrillas, J. Fer-
nandez-Fuertes, J. Seghatchian, L. Gutierrez, Platelet-derived bio-products:
classification update, applications, concerns and new perspectives, Transfus.
Apher. Sci. (2019) 102716.
[25] P. Everts, K. Onishi, P. Jayaram, J.F. Lana, K. Mautner, Platelet-rich plasma: new
performance understandings and therapeutic considerations in 2020, Int. J.
Mol. Sci. 21 (20) (2020).
A. Acebes-Huerta, P. Martínez-Botía, G. Carbajo-Argüelles et al.
[26] P. Harrison, P. Subcommittee on Platelet, The use of platelets in regenerative
medicine and proposal for a new classification system: guidance from the SSC
of the ISTH, J. Thromb. Haemost. 16 (9) (2018) 1895–1900.
[27] P.B. Szklanna, M.E. Parsons, K. Wynne, H. O’Connor, K. Egan, S. Allen, F. Ni
Ainle, P.B. Maguire, The platelet releasate is altered in human pregnancy, Pro-
teomics. Clin. Appl. 13 (3) (2019) e1800162.
[28] E. Anitua, R. Prado, G. Orive, Allogeneic platelet-rich plasma: at the dawn of
an off-the-shelf therapy? Trends Biotechnol. 35 (2) (2017) 91–93.
[29] P.B. Maguire, M.E. Parsons, P.B. Szklanna, M. Zdanyte, P. Munzer, M. Chatterjee,
K. Wynne, D. Rath, S.P. Comer, M. Hayden, F. Ni Ainle, M. Gawaz, Comparative
platelet releasate proteomic profiling of acute coronary syndrome versus stable
coronary artery disease, Front. Cardiovasc. Med. 7 (2020) 101.
[30] A.M. Ojea-Perez, A. Acebes-Huerta, T. Arias-Fernandez, L. Gutierrez,
M.C. Munoz-Turrillas, Implementation of a closed platelet-rich-plasma prepa-
ration method using the local blood bank infrastructure at the Principality of
Asturias (Spain): back to basic methodology and a demographics perspective
after 1 year, Transfus. Apher. Sci. (2019).
[31] A. Caiado, G. Ferreira-Dos-Santos, S. Goncalves, L. Horta, P. Soares Branco,
Proposal of a new standardized freeze-thawing technical protocol for leuco-
cyte-poor platelet-rich plasma preparation and cryopreservation, Cureus 12 (7)
(2020) e8997.
[32] M.E. Ritchie, B. Phipson, D. Wu, Y. Hu, C.W. Law, W. Shi, G.K. Smyth, limma
powers differential expression analyses for RNA-sequencing and microarray
studies, Nucleic. Acids. Res. 43 (7) (2015) e47.
[33] J. Fernandez-Fuertes, T. Arias-Fernandez, A. Acebes-Huerta, M. Alvarez-Rico,
L. Gutierrez, Clinical response after treatment of knee osteoarthritis with
a standardized, closed-system, low-cost platelet-rich plasma product: 1-year
outcomes, Orthop. J. Sports Med. 10 (3) (2022) 23259671221076496.
[34] I. van der Bijl, M. Vlig, E. Middelkoop, D. de Korte, Allogeneic platelet-rich
plasma (PRP) is superior to platelets or plasma alone in stimulating fibrob-
last proliferation and migration, angiogenesis, and chemotaxis as relevant pro-
cesses for wound healing, Transfusion 59 (11) (2019) 3492–3500.
[35] E.W. Raines, D.F. Bowen-Pope, R. Ross, Plasma binding proteins for
platelet-derived growth factor that inhibit its binding to cell-surface receptors,
Proc. Natl. Acad. Sci. U.S.A 81 (11) (1984) 3424–3428.
[36] M. Cattaneo, M. Chiodini, P.M. Mannucci, Ristocetin and platelet aggregation,
Br. J. Haematol. 71 (2) (1989) 301–302.
[37] Y. He, Y. Pei, K. Liu, L. Liu, Y. Tian, H. Li, M. Cong, T. Liu, H Ma, H You, J Jia,
D. Zhang, P. Wang, GITR/GITRL reverse signalling modulates the proliferation
of hepatic progenitor cells by recruiting ANXA2 to phosphorylate ERK1/2 and
Akt, Cell Death Dis 13 (4) (2022) 297.
[38] Z. Meng, G. Feng, X. Hu, L. Yang, X. Yang, Q.S.D.F. Jin, Factor-1α Promotes the
Migration, Proliferation, and Osteogenic Differentiation of Mouse Bone Marrow
Mesenchymal Stem Cells Through the Wnt/β -Catenin Pathway, Stem Cells Dev
30 (2) (2021) 106–117.
[39] J.R. Evanson, M.K. Guyton, D.L. Oliver, J.M. Hire, R.L. Topolski, S.D. Zumbrun,
J.C. McPherson, J.A. Bojescul, Gender and age differences in growth factor
concentrations from platelet-rich plasma in adults, Mil. Med. 179 (7) (2014)
799–805.
[40] H.S. Cho, I.H. Song, S.Y. Park, M.C. Sung, M.W. Ahn, K.E. Song, Individual vari-
ation in growth factor concentrations in platelet-rich plasma and its influence
on human mesenchymal stem cells, Korean J. Lab. Med. 31 (3) (2011) 212–218.
[41] Y. Taniguchi, T. Yoshioka, H. Sugaya, M. Gosho, K. Aoto, A. Kanamori, M. Ya-
mazaki, Growth factor levels in leukocyte-poor platelet-rich plasma and cor-
relations with donor age, gender, and platelets in the Japanese population, J.
Exp. Orthop. 6 (1) (2019) 4.
[42] S. Dangwal, B. Stratmann, C. Bang, J.M. Lorenzen, R. Kumarswamy, J. Fiedler,
C.S. Falk, C.J. Scholz, T. Thum, D. Tschoepe, Impairment of wound healing in
147
Acta Biomaterialia 177 (2024) 132–147
patients with type 2 diabetes mellitus influences circulating MicroRNA pat-
terns via inflammatory cytokines, Arterioscler. Thromb. Vasc. Biol. 35 (6)
(2015) 1480–1488.
[43] Y. Li, Y. Liang, Y. Gao, D. Chen, X. Ran, Dynamic changes of wound-related miR-
NAs after application of autologous platelet-rich gel in diabetic wounds, Chin.
Med. J. 135 (21) (2022) 2644–2646.
[44] C. Del Amo, A. Perez-Valle, L. Atilano, I. Andia, Unraveling the signaling secre-
tome of platelet-rich plasma: towards a better understanding of its therapeutic
potential in knee osteoarthritis, J. Clin. Med. 11 (3) (2022).
[45] M.E.M. Parsons, P.B. Szklanna, J.A. Guerrero, K. Wynne, F. Dervin, K. O’Connell,
S. Allen, K. Egan, C. Bennett, C. McGuigan, C. Gheveart, F. Ni Ainle, P.B. Maguire,
Platelet releasate proteome profiling reveals a core set of proteins with low
variance between healthy adults, Proteomics 18 (15) (2018) e1800219.
[46] M. Caceres, C. Martinez, J. Martinez, P.C. Smith, Effects of platelet-rich and
-poor plasma on the reparative response of gingival fibroblasts, Clin. Oral Im-
plants Res. 23 (9) (2012) 1104–1111.
[47] M. Shahidi, M. Vatanmakanian, M.K. Arami, F. Sadeghi Shirazi, N. Esmaeili,
S. Hydarporian, S. Jafari, A comparative study between platelet-rich plasma
and platelet-poor plasma effects on angiogenesis, Med. Mol. Morphol. 51 (1)
(2018) 21–31.
[48] F. Chellini, A. Tani, S. Zecchi-Orlandini, C. Sassoli, Influence of platelet-rich
and platelet-poor plasma on endogenous mechanisms of skeletal muscle re-
pair/regeneration, Int. J. Mol. Sci. 20 (3) (2019).
[49] C.E. Martinez, P.C. Smith, V.A. Palma Alvarado, The influence of platelet-derived
products on angiogenesis and tissue repair: a concise update, Front. Physiol. 6
(2015) 290.
[50] A.F. Drew, H. Liu, J.M. Davidson, C.C. Daugherty, J.L. Degen, Wound-healing de-
fects in mice lacking fibrinogen, Blood 97 (12) (2001) 3691–3698.
[51] C.T. Drinnan, G. Zhang, M.A. Alexander, A.S. Pulido, L.J. Suggs, Multimodal re-
lease of transforming growth factor-beta1 and the BB isoform of platelet de-
rived growth factor from PEGylated fibrin gels, J. Control Release 147 (2) (2010)
180–186.
[52] A.T. Nurden, P. Nurden, M. Sanchez, I. Andia, E. Anitua, Platelets and wound
healing, Front. Biosci. 13 (2008) 3532–3548.
[53] M. Lamkanfi, N. Festjens, W. Declercq, T. Vanden Berghe, P. Vandenabeele, Cas-
pases in cell survival, proliferation and differentiation, Cell Death Differ. 14 (1)
(2007) 44–55.
[54] Y. Akasaka, Y. Ishikawa, I. Ono, K. Fujita, T. Masuda, N. Asuwa, K. Inuzuka,
H. Kiguchi, T. Ishii, Enhanced expression of caspase-3 in hypertrophic scars and
keloid: induction of caspase-3 and apoptosis in keloid fibroblasts in vitro, Lab.
Invest. 80 (3) (20 0 0) 345–357.
[55] E. Anitua, M. de la Fuente, J. Merayo-Lloves, F. Muruzabal, G. Orive, Allogeneic
blood-based therapies: hype or hope? Eye 31 (4) (2017) 509–510.
[56] J.M. Burkhart, M. Vaudel, S. Gambaryan, S. Radau, U. Walter, L. Martens,
J. Geiger, A. Sickmann, R.P. Zahedi, The first comprehensive and quantitative
analysis of human platelet protein composition allows the comparative analy-
sis of structural and functional pathways, Blood 120 (15) (2012) e73–e82.
[57] J. Coppinger, D.J. Fitzgerald, P.B. Maguire, Isolation of the platelet releasate,
Methods Mol. Biol. 357 (2007) 307–311.
[58] H.E. Kattlove, B. Alexander, The effect of cold on platelets. I. Cold-induced
platelet aggregation, Blood 38 (1) (1971) 39–48.
[59] H.E. Kattlove, B. Alexander, F. White, The effect of cold on platelets. II. Platelet
function after short-term storage at cold temperatures, Blood 40 (5) (1972)
688–696.
[60] Y.M. Kuo, T.A. Kokjohn, W. Kalback, D. Luehrs, D.R. Galasko, N. Cheval-
lier, E.H. Koo, M.R. Emmerling, A.E. Roher, Amyloid-beta peptides interact
with plasma proteins and erythrocytes: implications for their quantitation in
plasma, Biochem. Biophys. Res. Commun. 268 (3) (20 0 0) 750–756.