European

E LucarelliCells
et al.and Materials Vol. 20 2010 (pages 13-23) DOI: 10.22203/eCM.v020a02 ISSN
A new platelet-rich 1473-2262
fibrin matrix

A RECENTLY DEVELOPED BIFACIAL PLATELET-RICH FIBRIN MATRIX

E. Lucarelli1*, R. Beretta2, B. Dozza1, P.L. Tazzari3, S.M. O’Connell4, F. Ricci3, M. Pierini1,
S. Squarzoni5, P.P. Pagliaro3, E.I. Oprita6, and D. Donati1

1
Bone Regeneration Laboratory, Dipartimento di Patologie Ortopediche-Traumatologiche Specialistiche, Rizzoli
Orthopaedic Institute, Bologna, Italy
2
Cascade Medical Enterprises LLC, Research and Development, Wayne NJ, USA
3
Service of Transfusion Medicine, S. Orsola-Malpighi Hospital, Bologna, Italy
4
Department of Vascular and General Surgery, Englewood Hospital & Medical Center, Mt. Sinai School of
Medicine, Englewood, NJ, USA
5
Institute of Molecular Genetics (IGM)-CNR, Unit of Bologna c/o Rizzoli Orthopaedic Institute, Bologna, Italy
6
National Institute R&D for Biological Sciences, Bucharest Romania
Abstract Introduction

Platelet-rich plasma (PRP) is used clinically in liquid or Platelets are known to release a variety of factors on
gel form to promote tissue repair. Because of the poor activation. These factors have a positive effect on tissue
mechanical properties, conventional PRP is often difficult repair. Platelets are easily collected from the blood stream
to handle when used in clinical settings and requires secure and are concentrated in a small volume of plasma known
implantation in a specific site, otherwise when released as platelet-rich plasma (PRP). PRP has been used in a
growth factors could be washed out during an operation. variety of surgical settings for soft-tissue healing
In this study, we analyzed the end product of a recently enhancement, including chronic lower extremity ulcer
developed commercially available system (FIBRINET®), healing (Mazzucco et al., 2004; O’Connell et al., 2008),
which is a dense pliable, platelet-rich fibrin matrix (PRFM). and tendon healing (Franchini et al., 2006; Gamradt et
We characterized the mechanical properties of PRFM and al., 2007; Maniscalco et al., 2008; Sánchez et al., 2007;
tested whether PRFM releases growth factors and whether Virchenko and Aspenberg, 2006). It is also used in
released factors induce the proliferation of mesenchymal orthopaedic and oral maxillofacial surgery where it
stem cells (MSC). Mechanical properties as well as platelet accelerates autogenous bone graft healing (Fennis et al.,
distribution were evaluated in PRFM. PRFM demonstrated 2004; Marx et al., 1998; Simon et al., 2009), and in
robust mechanical properties, with a tear elastic modulus cosmetic surgery (Man et al., 2001). PRP has also been
of 937.3 + 314.6 kPa, stress at a break of 1476.0 + 526.3 used in combination with mesenchymal stem cells (MSC)
kPa, and an elongation at break of 146.3 + 33.8 %. PRFM to promote bone regeneration (Hibi et al., 2006; Ito et al.,
maintained its mechanical properties throughout the testing 2006; Kitoh et al., 2004; Kitoh et al., 2007; Lucarelli et
process. Microscopic observations showed that the platelets al., 2003; Pieri et al., 2008) or mixed with fat cells for
were localized on one side of the matrix. Elevated levels of breast reconstruction, to correct painful, adherent scars,
PDGF-AA, PDGF-AB, EGF, VEGF, bFGF and TGF-β1 and to solve progressive hemifacial atrophy (Azzena et
were measured in the day 1-conditioned media (CM) of al., 2007; Cervelli et al., 2009a; Cervelli et al., 2009b;
PRFM and growth factor levels decreased thereafter. BMP2 Cervelli et al., 2009c; Cervelli et al., 2009d; Fulton, 2003).
and BMP7 were not detectable. MSC culture media Once injected or implanted, PRP is thought to release
supplemented with 20% PRFM-CM stimulated MSC cell growth factors locally for several days, inducing
proliferation; at 24 and 48 hours the induction of the accelerated tissue repair (O’Connell et al., 2006).
proliferation was significantly greater than the induction Currently, several methods and systems are available for
obtained with media supplemented with 20% foetal bovine the preparation of PRP, most producing a liquid end
serum. The present study shows that the production of a product. The physical properties of PRP can be changed
dense, physically robust PRFM made through high-speed if plasma and platelets are stimulated, usually by the
centrifugation of intact platelets and fibrin in the absence addition of CaCl2 and bovine thrombin, to produce a fibrin
of exogenous thrombin yields a potential tool for network. In this case, the end product is generally a weak
accelerating tissue repair. gel. The physical properties of fibrin gels have been
elucidated in previous studies. These studies have
Keywords: Platelet-rich fibrin matrix (PRFM), platelet-rich demonstrated that the use of different thrombin-fibrinogen
plasma (PRP), physical properties, fibrin, mesenchymal concentration ratios greatly affected the gel structure
stem cells. (Blomback and Okada, 1982; Blomback et al., 1989).
Other factors such as protein and ion concentrations
*Address for correspondence: (Chung and Shu, 1990; Okada et al., 1983; Okada et al.,
Enrico Lucarelli 1985), including calcium, also have an effect on gel
Bone Regeneration Laboratory, structure (Okada and Blomback, 1983). Among the
Dipartimento di Patologie Ortopediche-Traumatologiche commercially available systems, a recently developed
Specialistiche, Rizzoli Orthopaedic Institute, system has been developed (FIBRINET®) in which the
Via di Barbiano 1/10, I-40136, Bologna, Italy end product, called platelet-rich fibrin matrix (PRFM), is
Telephone Number: +39 051 6366595 produced with the combined use of gravitational force
FAX Number: +39 051 6366799 and calcium without the use of exogenous thrombin and
E-mail: enrico.lucarelli@ior.it appears physically stronger than conventional PRPs. In
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E Lucarelli et al. A new platelet-rich fibrin matrix

this study, we characterized the mechanical properties of microscopic observation. PRFM organization was assessed
PRFM and tested whether growth factors released from using the Bio-Rad MRC-1024 confocal microscope (Bio-
PRFM could support MSC proliferation, making it suitable Rad Laboratories, Hercules, CA, USA) equipped with a
for a variety of tissue engineering applications. krypton/argon laser. Incident wavelength was 568 nm for
Texas Red. Microphotographs were taken using a Focus
Imagecorder Plus (Focus Graphic Inc., St. Louis, MO,
Materials and Methods USA) on Kodak Film.

PRFM Preparation Scanning Electron Microscopy. Membrane samples were

PRFM was prepared according to the manufacturer’s cut into uniform shapes, washed with 0.1 M cacodylate
instructions (FIBRINET®, Cascade Medical Enterprises, buffer (pH 7.3) and fixed with 2.5% glutaraldehyde in 0.1
Wayne, NJ, USA). Briefly, 18 mL of human blood were M cacodylate buffer (pH 7.3) for 1 hour at 4°C. After a
collected into sterile supplied blood collection/separation second wash with 0.1 M cacodylate buffer (pH 7.3), the
tubes from healthy consenting blood donors (n=5). After samples were post-fixed with 1% osmium tetroxide (OsO4)
gentle mixing, the tubes were centrifuged at 1100g for 6 – 0.1 M cacodylate buffer for 1 hour at room temperature
minutes to obtain PRP. PRP was transferred into a calcium (22 ± 2°C). After rinsing with 0.1 M cacodylate buffer,
chloride-containing glass (0.25mL CaCl2 1M) bottle under the samples were dehydrated in an ethanol series and
sterile conditions. The bottle was gently swirled and then critical point dried. The samples were then mounted on
immediately placed in a swing-out rotor centrifuge aluminium stubs with silver adhesive paint, sputter-coated
(Heraeus GmbH, Hanau, Germany) and centrifuged at with 4 nm gold in an argon atmosphere in an Edwards
4500g for 25 minutes at 25°C. A translucent, yellow-white (Crawley, West Sussex, U.K.) S 150 B apparatus, and
platelet-fibrin matrix, having the same inner diameter as observed at 0° tilt angle with a Cambridge (Cambridge,
the bottle (33 mm) was recovered at the bottom. All of UK) Stereoscan 200 scanning electron microscope
these matrices were tested for tensile strength and elastic operated at 20 kV. For scanning electron microscopy, two
modulus five days after preparation. matrices were obtained from each of 18 mL of blood.

Blood Parameter Measurements Morphological Analysis. Average fibre diameter was

After informed consent venous blood was obtained from measured on digitized micrographs obtained by scanning
healthy donors, platelet counts were performed on the electron microscopy experiments. Each measure was taken
donors’ whole blood and on the residual serum remaining on 150 fibres on both sides of each membrane.
after PRFM preparation with a Coulter Haematology Subsequently, mean values and standard deviation (SD)
Analyzer (Beckman Coulter Inc., Brea, CA, USA). were calculated.
Fibrinogen concentration was measured with the Stago
Coagulation System employing the Clauss method (Roche, Mechanical Properties. To study the mechanical
Basel, Switzerland) on the same samples. For PRP, the properties of PRFM, specimens were fabricated by cutting
standard method was used (calibration for a linearity range out from each circular PRFM membrane a test specimen
from 100 to 600 mg/dL), while for serum, the ultra- in accordance to ASTMD 1708. All of these specimens
sensitive method was selected (calibration for a linearity were stored in sterile saline solution at 4°C and tested after
range from 1 to 50 mg/dL). These analyses were performed five days from preparation. All tests were performed at
on different samples with respect to the ones used for the 25°C. Tensile elastic modulus, elongation at break, and
mechanical tests. tensile strength (peak breaking energy) were evaluated
according to ASTM D 882:2209 and ASTM D 1708
Characterization of the PRFM standard procedures respectively, using an Instron
Immunofluorescence Confocal Microscopy. PRFM were (Norwood, MA, USA) 4302 mechanical testing station
cut into 5-mm squares for labelling with fluorescent with a loading cell of 10 N. Particular attention was paid
antibody (Ab) in order to investigate the tridimensional to fix the sample on the holder of the testing station. We
microscopic structure of the matrix using laser confocal used elastomeric tags to achieve secure, uniform grip of
microscopy. An indirect labelling procedure was used to every sample and to avoid possible damage to the strips.
fluorescently stain the membrane fibrin. The samples were
washed twice with a 10% glycine solution followed by PRFM Conditioned Media Collection
two more washes with 150 mM phosphate buffered saline Immediately following their preparation, PRFM prepared
(PBS) containing 1% bovine serum albumin (BSA). The from 3 different donors were placed in a 6-well plate where
samples were then incubated overnight with a solution of they were submerged in 3 ml of serum-free culture media
the primary antibody, Goat F(ab’)2 fragment specific to a-modified minimum essential medium (α-MEM, Sigma
human fibrinogen (ICN, Eschwege, Germany). The next Chemical, St. Louis, USA) and incubated in a humidified
day, the samples were washed for 1 hour in 20 mM PBS atmosphere at 37°C with 5% carbon dioxide. PRFM-
and 0.3% Triton X-100 and then treated with the secondary conditioned media (CM) containing the eluted growth
antibody (Donkey anti-goat Ab) labelled with Texas Red factors were collected in their entirety and an equal volume
(Jencons, Bedfordshire, UK). The samples were then of fresh serum-free α-MEM was added back into each well
washed thoroughly with 20 mM PBS and 0.3% Triton X- at 1, 2, 3 and 7 days. All collected CM samples were
100 and mounted with 90% glycerol on glass slides for divided into aliquots and immediately stored at -80°C until

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E Lucarelli et al. A new platelet-rich fibrin matrix

testing. CM were utilized to quantify growth factor Passage 3 MSC were seeded in 96-well plates (tissue
concentrations and to investigate their ability to promote culture grade, flat bottom) in serum-free α-MEM at an
MSC proliferation. initial density of 5 x 103 cells/well (that is, approximately
40% confluence). After overnight attachment, serum-free
Quantification of growth factors released from medium was removed and cells were cultured in α-MEM
PRFM supplemented 5%, 10% or 20% day 1 PRFM-CM. MSC
CM collected from all time points were assayed for platelet- maintained in normal cell culture medium (α-MEM
derived growth factor (PDGF)-AA, platelet-derived growth supplemented with 20% FBS) or serum free medium (α-
factor (PDGF)-AB, transforming growth factor (TGF) β- MEM) were used as positive and negative controls,
1, vascular endothelial growth factor (VEGF), epidermal respectively. Cell proliferation was assessed after 24, 48,
growth factor (EGF), basic fibroblast growth factor and 72 hours by methylene blue assay (Oliver et al., 1989).
(bFGF), bone morphogenetic protein (BMP)-2, and bone Briefly, cells were fixed by adding 100 μl of 10% formol
morphogenetic protein (BMP)-7. The growth factor saline to each well for 30 minutes. Cells were then stained
concentrations were assayed by quantitative enzyme-linked with 100 μl of filtered 1% (w/v) methylene blue in 0.01 M
immunosorbent assays (ELISA) (all human Quantikine® borate buffer (pH 8.5) added to each well. After 30 minutes,
ELISA kits were obtained from R&D Systems excess dye was removed. The remaining dye was then
(Minneapolis, MN, USA) according to manufacturer’s washed off with 0.01 M borate buffer (pH 8.5, 3x). After
instructions. Briefly, the samples and standards were the last rinse the cell layer still stained with methylene
prepared in duplicate according to the manufacturer’s blue was examined microscopically. The dye was then
protocol and added to 96 growth factor antibody coated eluted by adding 100 μl of 1:1 (v/v) ethanol and 0.1 M
well plates. The plates were incubated at room temperature, HCl to each well. The plates were then gently shaken and
washed, and incubated with selective enzyme-conjugated the absorbance at 655 nm (A655) measured for each well
antibodies for an additional time at room temperature. The by a microplate photometer (Synergy HT, BioTek
wells were then washed and substrate was added for 30 Instruments Inc.). The photometer was blanked on control
minutes at room temperature. Stop solution was added to wells containing elution solvent alone. Each treatment
each well, and the absorptions at 450 nm were determined condition was performed in quadruplicate. A linear
using a microplate photometer (Synergy HT, BioTek relationship between absorbance, determined in the assay,
Instruments Inc., Winooski, VT, USA). Standard curves and cell number was verified by counting the cells with a
for each growth factor were generated and growth factor NucleoCounter (Chemometec, Allerod, Denmark).
concentrations (pg/mL) of each sample were determined.
Samples were measured in appropriate dilutions prepared Statistical Analysis
as required to fit the respective calibration curves. The Results were determined from a minimum of three
minimum detection limits of these assay reported by the independent experimental data sets with duplicate
manufacturer are 2.3 pg/mL for PDGF-AA, 1.7 pg/mL for measurements per experiment. Data are expressed as mean
PDGF-AB, 4.6 pg/mL for TGFβ1, 5.0 pg/mL for VEGF, ± standard deviation. Statistical analysis was performed
0.7 pg/mL for EGF, 3.0 pg/mL for bFGF, 11 pg/mL for by one-way analysis of variance (ANOVA). When
BMP-2 e 2.4 pg/mL for BMP-7. significance was detected, post-hoc comparisons between
different groups were made using Bonferroni’s test for
Mesenchymal stem cell isolation, culture and growth multiple comparisons. A probability of less than 0.05 was
kinetic determination considered to be statistically significant. The statistical
Growth factors released from platelets have been shown program GraphPad - Prism4 (GraphPad Software, San
to promote MSC proliferation (Doucet et al., 2005; Diego, CA, USA) was used for the analysis.
Lucarelli et al., 2003). The ability of factors released from
PRFM to support proliferation of primary MSC cultures
was tested on MSC isolated from 3 patients undergoing Results
elective surgery at Rizzoli Orthopaedic Institute. Cultures
were established after obtaining informed consent and PRFM Description
according to a protocol approved by the Rizzoli’s Ethics To investigate the efficiency of the preparation procedure,
Committee. Briefly, a 10 mL bone marrow sample was we evaluated fibrinogen levels before and after matrix
aspirated from the anterior iliac crest. Mononuclear cells formation. Results shown in Table 1 demonstrate that no
were isolated in a density gradient and suspended in (α- detectable fibrinogen was left in the residual serum. These
MEM) containing 20% foetal bovine serum (FBS, results indicate that all the fibrinogen originally present
Euroclone, Wetherby, UK). All the nucleated cells were within the PRP was polymerized to fibrin in the PRFM. In
plated in 150 cm2 culture flasks and incubated in a addition, blood parametric analysis of the residual serum
humidified atmosphere at 37°C with 5% carbon dioxide. showed that only 0.9 % of platelets were left in the serum,
Non-adherent cells were discarded after 3 days, and indicating that 99.1% of the platelets were present in the
adherent cells were cultured for further expansion. When PRFM (Table 1). Platelet recovery data from PRP obtained
cultured flasks reached near confluence, cells were with the PRFM FIBRINET® kit have been previously
detached by mild trypsinization and reseeded onto new published from Leitner et al. being 65.5% (Leitner et al.,
flasks at one-third density for continued passage. Media 2006; Mazzucco et al., 2009). Considering the platelet
were changed every 3 to 4 days. concentration factor represented by the PRFM, use of the

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E Lucarelli et al. A new platelet-rich fibrin matrix

Table 1. Fibrinogen and Platelet Concentration in PRP, Whole Blood, and Residual Serum after Platelet-Rich Fibrin
Matrix Preparation (Residual Serum). Data are presented as mean ± standard deviation ( n=5).

Fibrinoge n conce ntration (mg/dL)

PRP 3 2 5 . 2 0 ± 4 7 . 42

Residual Serum Undetectable

Plate le t conce ntration (plate le t numbe r/mm3)

Whole Blood 299,000 ± 80,34 6

Residual Serum 2,600 ± 547.72** * ***

p<0.001.

Table 2. Mechanical Characteristics of PRFM. Tensile

PRFM kit produces a 210-fold higher concentration of
strength (stress and elongation at break) and elastic
platelets and fibrin when compared to the initial input
modulus were measured. Data are expressed as mean ±
whole blood volume.
standard deviation of three determination of five donors.
Macroscopically, the PRFM is a translucent yellow-
white disk (Fig. 1A) of 0.105 ± 0.021 mm thickness and PRFM Te ns ile Prope rtie s
33 mm diameter, with both sides of the membrane
appearing the same. PRFM is easy to handle and does not Stre s s at Elongation at Elas tic
tear when manipulated with forceps (Fig. 1B and 1C). We bre ak (KPa) Bre ak (%) M odulus (kPa)
observed that the PRFM produced by the FIBRINET® kit
appeared stable. Confocal microscopy was used to observe 1476 ± 526.3 146.3 ± 33.8 937.3 ± 314.6
fibrin architecture in PRFM labelled with a fluorescent
antibody against human fibrinogen (Fig. 2). PRFM
consisted of a very compact, coarse, fibrin network. On the bottom of the membrane, where fibres are
Scanning electron micrographs of the PRFM revealed that pressed against the flat surface of the glass container and
the two sides of the matrix are different. On one side, subjected to high centrifugal force stress, we can clearly
platelets are visible within the fibrin network (Fig. 3A and see two distinct populations: one showing sizes that are
3C, see Fig. 3D and 3F for higher magnification). In close to the native fibres with an average diameter of 95.7
particular there was a region (Fig. 3C and 3F) in which ± 29.5 nm; the second population has an average diameter
platelets and cells completely covered the fibrin network. of 305.3 ± 95.9 nm. This second population consists mainly
Platelets observed have mostly a lenticular shape that is of “pressed” bundles made of smaller fibres packed
consistent with an inactivated state. The larger cells longitudinally. Both populations are short fibres and
observed are consistent with residual white blood cells. bundles that are linked through a high density branch. On
On the reverse side of the matrix no platelets or cells were this lower side of the membrane, the formation of large
visible, only the fibrin network (Figs. 3B and 3E). The cables made of fused parallel fibres and bundles is even
morphological analysis of the fibrin network shows that, more pronounced. Large areas of the underside of the
on the upper side of the membrane, the fibres are organized membrane consist of fibre constructs that are strongly
in twisted parallel strands and bundles, frequently reaching pressed against the glass surface of the container forming
considerable diameters up to 1.1 μm. The average diameter dense aggregates, with no pores. The microscopic view
of isolated fibres in this side is close to what would be shows an alternating pattern of large strands and bundles
expected for a native fibrin clot, dehydrated and critical with obvious pores, and thick, non-porous areas, consisting
point-dried and analyzed with scanning electron of fused strands and amorphous fibrin (Fig. 3D). There is
microscopy (Collet et al., 2004; Collet et al., 2005). The also evidence of fibres and bundles with a kinked structure.
average diameter measured on this side of PRFM is 63.6 In general, both sides of the membrane demonstrated very
± 30.3 nm. high fibre density compared to native fibrin clots.

Fig. 1. (A) Macroscopic image of the PRFM. (B) PRFM before, and (C) after it is subjected to manual strain with a
forceps.

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E Lucarelli et al. A new platelet-rich fibrin matrix

Fig. 2. Confocal fluorescence

microscopic image of fibrin fibrils in a
PRFM highlighted by incorporation of
fluorescent-labelled indirect anti-
fibrinogen antibody system. The picture
was taken in a representative field. The
scale bar = 10 μm.

Fig. 3. Scanning electron micrograph images of the PRFM. (A) Representative picture obtained at the periphery of
the disk. (B) The reverse side of the membrane in which no platelets or cells are visible, but a coarse surface of fibrin
strands and bundles is visible. (C) Representative picture of the platelet/cell-rich area. (D) The same field as in A at
higher magnification shows platelets on the surface of the fibrin network. (E) A higher magnification of the same
field as in B of the reverse side of the membrane in which no platelets or cells are visible, but a coarse surface of fibrin
strands and bundles is visible. (F) A higher magnification of the same field as in C shows a thick layer of unactivated
platelets in which a few nucleated cells are visible.

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E Lucarelli et al. A new platelet-rich fibrin matrix

A B

C D

E F

Fig. 4. Quantification of growth factor release in the PRFM- conditioned media (CM) obtained from three donors.
Mean concentrations ± SD (n=3) of the indicated growth factors in the PRFM-CM at the indicated time point of CM
collection are presented. (A) PDGF AA, (B) PDGF-AB, (C) TGF-β1, (D) VEGF, (E) EGF and (F) bFGF.

Mechanical Properties day 1 compared to day 2, day 3 and day 7 (Fig. 4). These
Mechanical testing showed that PRFM has enhanced results are consistent with an abundant release of the
mechanical properties with respect to all the other platelet growth factors within the first day, and a gradual decrease
gels described in the literature, with a tear elastic modulus of the release of the growth factors thereafter. The kinetics
(expression of the stiffness) of 937.3 ± 314.6 kPa and stress of release varied among growth factors, release was
at break of 1476.0 ± 526.3 kPa, while the elongation at greatest within the first day for VEGF (Fig. 4D), while a
break reaches 146.3% ± 33.8 kPa (Table 2). Mechanical more gradual release was seen with bFGF (Fig. 4F). BMP-
properties of samples kept refrigerated in a saline solution 2 and BMP-7 were always below the level of detection.
for 18 days were not significantly different compared to
the ones measured after 5 days from preparation (data not MSC proliferation induced by PRFM-CM
shown). The results of the methylene blue assay performed on the
MSC obtained from three different donors subjected to
Growth factors released from PRFM different concentrations of day 1-PRFM-CM are shown
The concentration of PDGF-AA, PDGF-AB, TGF-β1, in Fig. 5. The increase in MSC proliferation of 5%, 10%
VEGF, EGF and bFGF in the PRFM-CM was greater at and 20% day 1-PRFM-CM was tested up to 72 hours.

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E Lucarelli et al. A new platelet-rich fibrin matrix

Fig. 5. PRFM-conditioned media (CM) induced cell proliferation. MSC proliferation was determined by methylene
blue assay after 24, 48, and 72 hours in culture. Mean optical density values displayed as % of seeded cells referred
as 100% (line). Data represent mean values ± SD of measurements obtained from MSC harvested from 3 donors.
CM were obtained from PRFM prepared from 3 donors. Each sample was assayed in quadruplicate. ** p < 0.01, ***
p < 0.001. Statistical significance was not reached (p > 0.05) where * for comparisons are not shown.

Optical density values indicated relative cell proliferation. absence of exogenous thrombin and centrifuged for the
Compared to the serum-free control, a statistically second time at high gravitational force to form the PRFM.
significant difference was observed at all time points and PRFM prepared with FIBRINET® system has been
with all concentrations of PRFM-CM tested (Fig. 5). These used previously to treat difficult lower-extremity ulcers
data indicate that factors released from the PRFM are active as described by O’Connell and co-workers (O’Connell et
and sustain the proliferative potential of MSC. The extent al., 2006; O’Connell et al., 2008). Possible reasons for the
of proliferation of MSC cultured with medium success of PRFM in closing these difficult ulcers may be
supplemented with 5% day 1-PRFM-CM up to 72 hours due to its mechanical properties, its localized high platelet
was significantly lower from the proliferation observed in concentration and its elevated cytocompatibility as a result
MSC supplemented with 20% FBS. Medium supplemented of its being produced directly from the patient’s own blood
with 10% of day 1-PRFM-CM support MSC proliferation without any exogenous organic additives. The results of
as 20% FBS up to 48 hours, while the effect significantly the studies described here show that the mechanical
decreased after 72 hours. MSC proliferation was properties of an autologous platelet-fibrin preparation can
significantly increased by 20% day1-PRFM-CM compared be improved by inducing extensive fibrin network
to 20% FBS up to 48 hours, while the effects of the two formation through increased gravitational force in the
supplemented media were not significantly different after second centrifugation during PRFM preparation. With this
72 hours. procedure, PRP can be produced in the form of a PRFM.
Conventional PRPs are usually liquids or weak gels, and
fibrin-based clots present an elastic modulus, or stiffness,
Discussion measured in the range of 0.1 to 1500 Pa, depending on the
clot structure and the physiologic conditions (Bale et al.,
Conventional PRP is usually produced during a two-step 1985; Carr et al., 1995; Collet et al., 2004; Collet et al.,
procedure (Fennis et al., 2004; Marx et al., 1998; 2005; Cummings et al., 2004; Urech et al., 2005; Weisel,
Mazzucco et al., 2004; Okada et al., 1983; Sánchez et al., 2004). In contrast, PRFM made of the same components
2007). In the first step, PRP is formed by separation of a in our experiments, gives an elastic modulus of 937.3 kPa.
platelet concentrate from the platelet poor plasma and the This level is comparable with that of arterial tissue (Zhang
white and red cell fraction. In the second step, exogenous et al., 2002), and represents approximately 50% of the
thrombin, or other activator such as bathroxobin stiffness of intact human skin (Zhang et al., 2007). These
(Mazzucco et al., 2008) is added together with calcium data indicate that PRFM is more than 600 times stiffer
chloride, or calcium gluconate, to the platelet concentrate than the stiffest fibrin clot obtained at ambient pressure
and the platelet poor plasma. This converts fibrinogen into and described in the literature to date.
fibrin, and the fibrin network is formed. In contrast, the In routine clinical practice, the improved mechanical
PRFM system isolates plasma and platelets in the first properties of PRFM over conventional PRP translate into
centrifugation using the thixotropic separator gel in the a biologic matrix that is easy to handle and implant in a
first tube. In the second step, PRP is re-calcified in the wide variety of tissue repair applications. The increased

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E Lucarelli et al. A new platelet-rich fibrin matrix

elastic modulus of the PRFM confers significant pliability the bottom of the container. In fact, each membrane
and drapability which, in addition to its increased tensile analysed shows a different distribution of platelets on
strength, allows the membrane to closely conform to a wide bottom side of the PRFM. It seems that the platelets tend
variety of irregular surgical sites and surfaces in a manner to lie on the lower level where they are pressed by the
similar to split-thickness skin autografts. Moreover, the centrifugal force.
matrix can easily be sutured to securely maintain contact We included the in vitro studies of the quantification
with the implanted site. This makes the PRFM preferable of growth factors released from PRFM and their ability to
for use in a clinical setting in which PRP has to be promote the growth of MSC to further support the
implanted in a specific site or where released growth factors “potential” of PRFM to accelerate tissue repair.
could be washed out during an operation as in arthroscopic Quantification of growth factor levels and the kinetics of
joint repair procedures (Maniscalco et al., 2008). release show similar concentrations of bFGF, VEGF, EGF
Currently available forms of autologous PRP are and TGF-β1 and similar release kinetics with previous
usually fragile, unstable, and prone to rapid fibrinolysis published results (Mazzucco et al., 2009). This is
and dissolution following implantation. This limitation not particularly remarkable considering that the method used
only prevents easy handling in a clinical setting but also to obtain the conditioned media was different in that the
when the PRP gel is to be used in association with isolated FIBRINET® kit has been used to produce a matrix gel
cells. We have shown that the mechanical properties of (Mazzucco et al., 2009) while in our study it has been
PRFM are robust and long lasting and that the PRFM is used to produce a membrane. Further studies are needed
effective in supporting MSC, proliferation, and survival. to investigate whether the matrix gel and the membrane
Scanning electron microscopy and confocal fluorescence release growth factors with a similar kinetics.
microscopy of PRFM show a strongly enhanced average Our results further show that growth factors released
fibre dimension, on the bottom side, compared to native from PRFM can support MSC proliferation. This is
fibrin gels described in literature (Blomback and Okada, consistent with published results that show that growth
1982, Blomback et al., 1989; Blomback et al., 1990; Muller factors released from platelets have the potential to
et al., 1984;Okada et al., 1983; Okada and Blomback, stimulate MSC proliferation (Doucet et al., 2005; Lucarelli
1983; Okada et al., 1985; Collet et al., 2004; Collet et al., et al., 2003). This finding is relevant in that it supports a
2005), with a remarkable increase to large strands, bundles therapeutic role of the PRFM alone if implanted in a site
and cables. The effect of branching is clearly evident on in which local MSC could be recruited to heal the damaged
this side of the membrane. At this point, the large bundles tissue. However, further studies will be needed to clarify
and cables appear to be compressed to the bottom of the whether MSC grown in PRFM keep the characteristics
container where the centrifugal force is maximal during and phenotype of MSC such as multi-lineage
PRFM preparation. For this reason, the fibres are strongly differentiation pluripotency and maintenance of MSC
deformed compared to their natural shape. In contrast, on markers. In vivo experiments employing PRFM itself or
the upper side of the matrix the texture appears to be coarse, together with MSC will be needed to confirm the
with a lot of twisted and bended fibres arranged in large, conclusion that PRFM accelerates tissue repair by itself
coiled bundles, frequently aggregated in long cables. This or associated with cells.
arrangement could contribute to the strongly enhanced In conclusion, production of a dense, cross-linked,
mechanical properties of this fibrin-based material, physically robust PRFM made of intact platelets and fibrin
specifically the elastic modulus and the elongation at break. by high-speed centrifugation in the absence of exogenous
More studies are needed to clarify the relationship between thrombin, yields an ideal scaffold for use in tissue repair
fibrinogen concentration, membrane thickness, platelet by itself (O’Connell et al., 2006; O’Connell et al., 2008;
concentration and the mechanical properties of the PRFM. Maniscalco et al., 2008) or in conjunction with exogenous
The algorithm linking centrifugal force applied to PRP, or viable cells. PRFM produced by the FIBRINET® system
to PPP, and mechanical properties of resulting fibrin is so different in makeup and physical characteristics as to
constructs are worth investigation as well. We also be significantly distinct and separate from conventional
observed that it is possible that the difference seen in fibre platelet-rich plasmas. PRFM can be easily tailored to the
size across the two sides of the matrix could be due to the desired shape by altering the shape of the second container.
delay of the coagulation process. The 25 minute In these studies we employed a glass bottle as second
centrifugation at 4500g, at 25°C, needed to prepare the container to produce a disk-shaped membrane. It is
PRFM may not be enough for fibrin synthesis and cross- interesting to note that with this shape, speed and duration
linking to go to completion. This would account for the of centrifugation, most of the platelets concentrate in one
smaller fibres found on the upper surface overlaying the side of the membrane. This sidedness may have clinical
more densely structured fibres found on the bottom layer implications for enhanced tissue repair. It would also apply
after high-speed centrifugation. To clarify this finding, new to isolated cell preparations, such as MSC or other cell
studies are currently underway. types added to the PRFM. Further studies using a larger
The fact that the platelets are grouped together in some number of donors are currently underway to confirm the
areas on bottom side of the membrane, instead of being viability, growth factor release and biological response of
evenly distributed on the membrane’s entire surface, is the platelets within and released from PRFM.
probably due to the particular conformation of the glass at

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E Lucarelli et al. A new platelet-rich fibrin matrix

Acknowledgements Collet JP, Nagaswami C, Farrell DH, Montalescot G,

Weisel JW (2004) Influence of fibrinogen splice variant
The authors are grateful to Ms. P. Dimopoulou and Cristina on fibrin physical properties and fibrinolysis rate.
Ghinelli for editorial assistance and to Dr. Sergio Lodi and Arterioscler Thromb Vasc Biol 24: 382-386.
Prof. Pietro Maniscalco for their invaluable assistance and Collet JP, Moen JL, Veklich YI, Gorkun OV, Lord SL,
advice during the preparation of this paper. We are grateful Montalescot G, Weisel JW (2005) The aC domains of
to Cascade Medical Enterprises, LLC, for donating the fibrinogen affect the structure of the fibrin clot, its
FIBRINET® kits for our study and for support for the mechanical properties, and its susceptibility to fibrinolysis.
mechanical testing and the confocal microscopy. Blood 106: 3824-3830.
Cummings CL, Gawlitta D, Nerem RM, Stegemann
Conflict of Interest JP (2004) Properties of engineered vascular constructs
made from collagen, fibrin, and collagen–fibrin mixtures.
Dr. S. O’Connell is a shareholder and receives monetary Biomaterials 25: 3699-3706.
compensation as a consultant of Cascade Medical Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy
Enterprises. Dr. R. Beretta is a shareholder of Cascade X, Lataillade JJ (2005) Platelet lysates promote
Medical Enterprises. mesenchymal stem cell expansion: a safety substitute for
animal serum in cell-based therapy applications. J Cell
Physiol 205: 228-236.
Fennis JP, Stoelinga PJ, Jansen JA (2004) Mandibular
References reconstruction: a histological and histomorphometric study
on the use of autogenous scaffolds, particulate cortico-
Azzena B, Mazzoleni F, Abatangelo G, Zavan B, cancellous bone grafts and platelet rich plasma in goats.
Vindigni V (2007) Autologous platelet-rich plasma as an Int J Oral Maxillofac Surg 33: 48-55.
adipocyte in vivo delivery system: case report. Aesthetic Franchini M, Rocca G, Bosinelli A, Governa M, Olzer
Plast Surg 32: 155-161. D, Fiorini S, Aloisi O, Scalvi A, Gandini G, Barisoni D,
Bale MF, Muller MF, Ferry JD (1985) Rheological Marcer M, Aprili G (2006) Use of platelet gel and bone
studies of creep and creep recovery of unligated fibrin clots: from tissue bank in regenerative medicine as a
comparison of clots prepared with thrombin and ancrod. multidisciplinary model. Blood Transfus 4: 296-310.
Biopolymers 24: 461-482. Fulton JE (2003) Breast contouring with “gelled”
Blomback B, Okada M (1982) Fibrin gel structure autologous fat: a 10-year update. Intl J Cosm Surg Aes
and clotting time. Thromb Res 25: 51-70. Dermatol 5: 155-163.
Blomback B, Carlsson K, Hessel B, Liljborg A, Procyk Gamradt SC, Rodeo SA, Warren RF (2007) Platelet
R, Aslund N (1989) Native fibrin gel networks observed rich plasma in rotator cuff repair. Techniques in
by 3D microscopy, permeation and turbidity. Biochim Orthopaedics 22: 26-33.
Biophys Acta 997: 96-110. Hibi H, Yamada Y, Kagami H, Ueda M (2006)
Carr ME, Carr SL, Merten SR (1995) Effects on ionic Distraction osteogenesis assisted by tissue engineering in
and non-ionic contrast media on clot structure, platelet an irradiated mandible: a case report. Int J Oral Maxillofac
function and thrombolysis mediated by tissue plasminogen Implants 21: 141-147.
activator plasma clots. Haemostasis 25: 172-181. Ito K, Yamada Y, Naiki T, Ueda M (2006) Simultaneous
Cervelli V, Gentile P, Grimaldi M (2009a) Regenerative implant placement and bone regeneration around dental
surgery: Use of fat grafting combined with platelet-rich implants using tissue-engineered bone with fibrin glue,
plasma for chronic lower- extremity ulcers. Aesthetic Plast mesenchymal stem cells and platelet-rich plasma. Clin Oral
Surg 33: 340-345. Implants Res 17: 579-586.
Cervelli V, Gentile P, Grimaldi M (2009b) Use of cell Kitoh H, Kitakoji T, Tsuchiya H, Mitsuyama H,
fat mixed with platelet gel in progressive hemifacial Nakamura H, Katoh M, Ishiguro N (2004) Transplantation
atrophy. Aesthetic Plast Surg 33: 22-27. of marrow-derived mesenchymal stem cells and platelet-
Cervelli V, Gentile P, Scioli MG, Grimaldi M, Spagnoli rich plasma during distraction osteogenesisa preliminary
LG, Orlandi A (2009c) Application of platelet-rich plasma result of three cases. Bone 35: 892-898.
to fat grafting during plastic surgical procedures: Clinical Kitoh H, Kitakoji T, Tsuchiya H, Katoh M, Ishiguro N
and in vitro evaluation. Tissue Eng Part C Methods 15: (2007) Distraction osteogenesis of the lower extremity in
625-634 patients with achondroplasia/hypochondroplasia treated
Cervelli V, Palla L, Pascali M, De Angelis B, Curcio with transplantation of culture-expanded bone marrow
BC, Gentile P (2009d) Autologous platelet-rich plasma cells and platelet-rich plasma. J Pediatr Orthop 27: 629-
mixed with purified fat graft in aesthetic plastic surgery. 634.
Aesthetic Plast Surg 33: 716-721. Leitner GC, Gruber R, Neumüller J, Wagner A,
Chung YL, Shu C (1990) Fibrinogen, thrombosis, Kloimstein P, Höcker P, Körmöczi G, Buchta C (2006)
coagulation and fibrinolysis. In: Native Fibrin Gel Platelet content and growth factor release in platelet-rich-
Networks and Factors Influencing Their Formation in plasma: a comparison of four different systems. Vox Sang
Health and Disease (Blomback B, Carlsson K, Banerjee 91: 135-139.
D, Hamsten A, Hessel B, Procyk R, Silveira A, Zacharski Lucarelli E, Beccheroni A, Donati D, Sangiorgi L,
L, eds.). Plenum Press New York, London, pp. 1-23. Cenacchi A, Del Vento AM, Meotti C, Zambon Bertoja A,

www.ecmjournal.org
21
E Lucarelli et al. A new platelet-rich fibrin matrix

Giardino R, Fornasari PM, Mercuri M, Picci P (2003) Simon BI, Zatcoff AL, Kong JJW, O’Connell SM
Platelet-derived growth factors enhance proliferation of (2009) Clinical and histological comparison of extraction
human stromal stem cells. Biomaterials 24: 3095-3100. socket healing following the use of Autologous platelet-
Man D, Plosker H, Winland-Brown JE, Saltz R (2001) rich fibrin matrix (PRFM) to ridge preservation procedures
The use of autologous platelet-rich plasma (platelet gel) employing demineralized freeze dried bone allograft
and autologous platelet-poor plasma (fibrin glue) in material and membrane. Open Dent J 3: 92-99.
cosmetic surgery. Plast Reconstr Surg 107: 238-239. Urech L, Bittermann AG, Hubbell JA, Hall H (2005)
Maniscalco P, Gambera D, Lunati A, Vox G, Mechanical properties, proteolytic degradability and
Fossombroni V, Beretta R, Crainz E (2008) The “Cascade” biological modifications affect angiogenic process
membrane: a new PRP device for tendon ruptures. extension into native and modified fibrin matrices in vitro.
Description and case report on rotator cuff tendon. Acta Biomaterials 26: 1369-1379.
Biomed 79: 223-226. Virchenko O, Aspenberg P (2006) How can one platelet
Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, injection after tendon injury lead to a stronger tendon after
Strauss JE, Georgeff KR (1998) Platelet-rich plasma: 4 weeks? Interplay between early regeneration and
Growth factor enhancement for bone grafts. Oral Surg Oral mechanical stimulation. Acta Orthop 77: 806-812.
Med Oral Pathol Oral Radiol Endod 85: 638-646. Weisel JW (2004) The mechanical properties of fibrin
Mazzucco L, Medici D, Serra M, Panizza R, Rivara G, for basic scientists and clinicians. Biophys Chem 112: 267-
Orecchia S, Libener R, Cattana E, Levis A, Betta PG, 276.
Borzini P (2004) The use of autologous platelet gel to treat Zhang D, Eggleton CD, Arola DD (2002) Evaluating
difficult-to-heal wounds: a pilot study. Transfusion 44: the mechanical behaviour of arterial tissue using digital
1013-1018. image correlation. Exp Mech 42: 409-416.
Mazzucco L, Balbo V, Cattana E, Borzini P (2008) Zhang GA, Ning FG, Zhao NM (2007) Biomechanical
Platelet-rich Plasma and Platelet gel preparation properties of four dermal substitutes. Chinese Med J 120:
Plateltex®. Vox Sang 94: 202-208. 1454-1455.
Mazzucco L, Balbo V, Cattana E, Guaschino R, Borzini
P (2009) Not every PRP is born equal. Evaluation of growth
factor availability for tissue through our PRP-gel Discussion with Reviewers
preparations: FIBRINET®, RegenPRP-kit®, Plateltex®,
and one manual procedure. Vox Sang 97: 110-118. Reviewer I: Given the interesting mechanical properties
Muller FM, Ris H, Ferry JD (1984) Electron and their release of growth factors (PDGF, VEGF, EGF,
microscopy of fine fibrin clots and fine and coarse fibrin BMP-2 especially), what do you expect from the
films. J Mol Biol 174: 369-384. engraftment and in vivo performance of PRFM, alone or
O’Connell SM, Impeduglia T, Hessler K, Wang X, loaded with cells? Which clinical applications would be
Carroll RJ, Dardik H (2006) Autologous platelet-rich fibrin the most obvious for such a material?
matrix as a stimulator of healing of chronic lower extremity Authors: The growth factors released by platelets in
ulcers. Wound Rep Regen 14: A76. response to stimulation (EGF, PDGF-AA and BB, IGF-I
O’Connell SM, Impeduglia T, Hessler K, Wang XJ, and II, bFGF, VEGF, TGF-β 1 and 2, etc) are the same as
Carroll RJ, Dardik H (2008) Autologous platelet-rich fibrin those involved in the initial phases of repair of most, if not
matrix as cell therapy in the healing of chronic lower- all tissues of the body. Providing them in a concentrated
extremity ulcers. Wound Rep Regen 16: 749-755 biologic form, in their normal ratios (as opposed to
Okada M, Blomback B (1983) Calcium and fibrin gel excessive concentrations of a single growth factor) and
structure. Thromb Res 29: 269-280. capable of gradual release to the repair site over days and
Okada M, Blomback B, Block M (1983) Effect of weeks would serve to augment healing and repair in a wide
albumin and dextran on fibrin gel structure. Thromb variety of clinical applications. This would be particularly
Haemost 50: 201 (Abstr 613). true in cases where healing is compromised (i.e., poor
Okada M, Blomback B, Chang MD, Horowitz B (1985) perfusion/ischemia, repeated trauma, large/critical-size
Fibronectin and fibrin gel structure. J Biol Chem 260: 1811- defects, underlying systemic pathology, etc.). As to
1820. engraftment, clinical experience in the treatment of lower
Oliver MH, Harrison NK, Bishop JE, Cole PJ, Laurent extremity ulcers indicates that the membrane persists for
GJ (1989) A rapid and convenient assay for counting cells 1 to 2 weeks before being absorbed in the healing wound
cultured in microwell plates: application for assessment bed (O’Connell et al., 2008). This is a fibrin matrix with
of growth factors. J Cell Sci 92: 513-518. intact platelets as opposed to a collagen matrix with living,
Pieri F, Lucarelli E, Corinaldesi G, Iezzi G, Piattelli A, proliferatively competent cells such as an autograft. As
Giardino R, Bassi M, Donati D, Marchetti C (2008) such the PRFM could be considered more of a cell therapy
Mesenchymal stem cells and platelet-rich plasma enhance than a viable graft capable of ‘take’. Clinical applications
bone formation in sinus grafting: a histomorphometric that can benefit from the PRFM therapy are many. In
study in minipigs. J Clin Periodontol 35: 539-546. addition to difficult to heal leg ulcers, other potential
Sánchez M, Anitua E, Azofra J, Andía I, Padilla S, applications include orthopaedic applications such as
Mujika I (2007) Comparison of surgically repaired Achilles rotator-cuff repair, ligament repair (ACL, PCL, etc.),
tendon tears using platelet-rich fibrin matrices. Am J Sports lumbar spine fusion, cranial maxillofacial reconstructive
Med 35: 245-251. surgery (large sinus lifts, alveolar defect reconstruction,

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E Lucarelli et al. A new platelet-rich fibrin matrix

prognathic maxillar and mandibular reconstruction), general, we found the frequency of membranes with poor
pressure ulcers, long bone non-union, etc. In short, the mechanical properties to be less than 1 in 200. The most
PRFM membrane could be of great potential benefit in frequent cause of membranes with poor characteristics was
both soft tissue and bony repair where surface are coverage concomitant drug therapy that interfered with the
and augmented healing is critical. Concerning the question, coagulation process, for example patients on maintenance
when it is appropriate to add stem cells or other cells to aspirin, Plavix, low-molecular weight heparin or other
PRFM, much more clinical work needs to be done. anticoagulant therapy. This is an area of continual interest
However, one obvious type of application would be in and surveillance to determine the extent and cause of inter-
those situations where there are insufficient viable cells patient variability in the production and clinical application
and tissue at or near the repair site for the PRFM to of PRFM.
stimulate. Examples of such applications could include
full thickness wounds such as large third degree burns, Reviewer II: One of the interesting properties of the PRP
traumatic wounds or ulcers that extend to exposed tendon for tissue engineering (added to the growth factors release
and bone. This would also be expected with critical-volume etc) is that it can be mixed with cells of interest and injected
bone defects where scaffold materials (demineralized bone before jellification. Obviously, this cannot be the case with
matrix, tri-calcium phosphate/hydroxy-apatite, etc.) and Fibrinet®, so it could only be used through invasive
stem cells would be useful additions to the PRFM. procedure. In addition, only materials of small size could
be produced, I guess. I would be interested in having your
Reviewer I: The authors describe a major heterogeneity opinion on the foreseen clinical applications.
of the fibrin network throughout the material. What could Authors: One of the distinctive characteristics of the
be the advantages and disadvantages of this heterogeneity Membrane kit is, that it has enhanced mechanical
in therapeutic applications? properties compared to PRP products obtained with
Authors: We maintain that the exceptional mechanical commercially available devices or manual procedures;
characteristics of the PRFM are directly related to the therefore, it does not produce an injectable material.
ultrastructure of each membrane. The presence of large However, the final product has dimensions that are
twisted bundles and cables, mixed with bent fibres and compatible with mini-invasive and arthroscopic
fused fibrin structures, is the basis of this extraordinary orthopaedic surgical procedures, where it is delivered and
elastic modulus and as such, is close to that of a thin latex anchored directly to the defect. A project is being carried
membrane. Concerning inter-patient variation, several out to manufacture a plastic disposable cartridge to produce
years of clinical development experience using the PRFM a square-shaped PRFM of considerably larger size at the
has demonstrated minimal inter-patient variability with point of care. For example: from 20 mL of PRP we can get
respect to mechanical properties and size of the membrane a 5x5 cm square membrane. In another project we planned
construct. The experience of a number of independent to produce a large size membrane to be stored in the
clinical investigators has been that gels and membranes refrigerator for many months, in sterile packages, to be
made from this autologous fibrin material are very reliable used when needed. This would be produced with the help
for use in a variety of different surgical applications. In of a specially-designed axial centrifuge.

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