Original Article

Journal of Biomaterials Applications

0(0) 1–13
Hemostatic wound dressings: Predicting ! The Author(s) 2019
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DOI: 10.1177/0885328219831095
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Cornelia Wiegand1 , Martin Abel2, Uta-Christina Hipler1,

Peter Elsner1, Michael Zieger3, Julia Kurz4, Hans P Wendel4 and
Sandra Stoppelkamp4

Abstract
Background: Application of controlled in vitro techniques can be used as a screening tool for the development of new
hemostatic agents allowing quantitative assessment of overall hemostatic potential.
Materials and methods: Several tests were selected to evaluate the efficacy of cotton gauze, collagen, and oxidized
regenerated cellulose for enhancing blood clotting, coagulation, and platelet activation.
Results: Visual inspection of dressings after blood contact proved the formation of blood clots. Scanning electron
microscopy demonstrated the adsorption of blood cells and plasma proteins. Significantly enhanced blood clot formation
was observed for collagen together with b-thromboglobulin increase and platelet count reduction. Oxidized regener-
ated cellulose demonstrated slower clotting rates not yielding any thrombin generation; yet, led to significantly increased
thrombin-anti-thrombin-III complex levels compared to the other dressings. As hemostyptica ought to function without
triggering any adverse events, induction of hemolysis, instigation of inflammatory reactions, and initiation of the innate
complement system were also tested. Here, cotton gauze provoked high PMN elastase and elevated SC5b-9
concentrations.
Conclusions: A range of tests for desired and undesired effects of materials need to be combined to gain some degree
of predictability of the in vivo situation. Collagen-based dressings demonstrated the highest hemostyptic properties with
lowest adverse reactions whereas gauze did not induce high coagulation activation but rather activated leukocytes
and complement.

Keywords
Blood clotting, coagulation, collagen, hemocompatibility, oxidized regenerated cellulose

Introduction
Basic treatment for bleeding wounds consists of absor-
bent dressings combined with efforts to stop blood
flow, such as pressure, wound packing, binding, etc.1
Hemostasis normally occurs in the order of minutes
with the exception of severe wounds or bleeding disor-
ders. During hemostasis, the damaged tissue space is
rapidly filled with a blood clot stopping fluid loss and
re-establishing a barrier to the outside. In the subse-
quent phases of healing, this clot will serve as a
temporary matrix for the cells that migrate toward
the wound to reconstruct the dermal tissue.2 Topical
hemostatic agents can induce or accelerate the coagu-
lation cascade. Different types of hemostatic agents are
available, including plant-derived (i.e. cellulose, poly-
saccharides), marine-derived (i.e. chitosan, alginate),
synthetic (i.e. polyethylene glycol, glutaraldehyde),
animal-derived (i.e. gelatin, collagen, albumin), and
human-derived (i.e. thrombin, fibrinogen) ones.3
Local hemostats are beneficial as they improve blood
conservation by reducing fluid loss, shorten the time
of hemostasis, avoid the adverse effects of systemic
1
Department of Dermatology, University Hospital Jena, Jena, Germany
2
Lohmann & Rauscher GmbH & Co. KG, Neuwied, Germany
3
SRH Wald-Klinikum Gera GmbH, Gera, Germany
4
Department of Thoracic, Cardiac and Vascular Surgery, University
Hospital Tuebingen, Tuebingen, Germany
Corresponding author:
Cornelia Wiegand, Department of Dermatology, University Hospital
Jena, Erfurter Str. 35, Jena 07743, Germany.
Email: c.wiegand@med.uni-jena.de
2 Journal of Biomaterials Applications 0(0)
hemostatic drugs, and save transfusion blood.4
Nonetheless, adverse effects such as foreign body reac-
tion, infection, and granuloma formation have been
reported.5 The ideal hemostatic material should be
easy to apply and remove, feature a high bioresorption
potential, and suture ability as well as exhibit low anti-
genicity and tissue reactivity effects.6 The possibility to
adjust absorbability and flexibility of the material
would further be advantageous.7 Cotton gauze, in the
form of pads or bandages, is the classic absorbent
dressing material. In these forms, it neither initiates
nor potentiates platelet activation or clot formation.1
Recently, some cotton materials have been described to
exhibit hypercoagulant activity depending on their
processing.8,9 Oxidized regenerated cellulose (ORC) is
a chemically altered form of cellulose. It has been
found to be more valuable than the standard cotton
gauze in dealing with bleeding as ORC seems to
confer hemostasis by decreasing the pH, acting as a
caustic generating an artificial clot, and interacting
with platelets as well as proteins of the intrinsic and
extrinsic pathway.10 However, the most widely used
topical hemostatic agents nowadays are collagen and
gelatin.4 The body’s collagen is a natural coagulant and
plays an important role in hemostasis and thrombo-
genesis.11 Collagen sponges as dressing material have
the ability to absorb large quantities of tissue exudate,
to smoothly adhere to the wound bed, to preserve a
moist climate, and to protect against mechanical
harm and secondary bacterial infection.12
Considerable effort has gone into developing
advanced dressings that are more than simply absor-
bent but actively cause or potentiate hemostasis.1 Yet,
it is difficult to judge them objectively and make com-
parisons of effectiveness as such a claim requires evi-
dence of platelet activation and aggregation and/or
fibrin formation.1,13 The application of controlled in
vitro techniques might serve as a screening tool in the
development of new hemostatic agents6 and allow
quantitative assessment of the overall hemostatic
potential of wound dressings in vitro. We have selected
several in vitro tests to evaluate the efficacy of diverse
dressing materials, such as cotton gauze, collagen, and
ORC, to enhance blood clotting and coagulation as
well as platelet activation. Therefore, thrombus forma-
tion on the dressing samples after blood contact was
inspected visually. Platelet adhesion and aggregation,
cell adhesion, and fibrinogen adsorption on the test
specimen were further evaluated by scanning electron
microscopy (SEM). Blood clot formation was detected
by photometric measurement of free hemoglobin and
assessed by calculation of the blood clotting index
(BCI). Material contact to blood initiates intrinsic
coagulation via factors XII, XI, and eventually factor
X which modulates prothrombin conversion.
Measurement of the thrombin-anti-thrombin-III com-
plex (TAT) served as a marker for thrombin generation
after contact to the dressing samples. Additionally, the
plasmatic thrombin generation was measured in vitro
using a commercially available thrombin generation
test, and the parameters peak thrombin, lag-time, and
time-to-peak were determined. For evaluation of plate-
let activation by the dressing samples, the concentra-
tion of b-thromboglobulin (b-TG) was measured.
Material contact to blood can further cause inflamma-
tory reactions and immune responses. Therefore,
hemocompatibility was evaluated by determining
hemolytic effects on erythrocytes and release of PMN
elastase from neutrophil granulocytes. In addition,
activation of the complement system was investigated.
Therefore, complement convertase C5a activity after
contact to the dressing materials and SC5b-9 concen-
trations, which serve as a marker for activation of the
terminal complement complex, were measured.
Materials and methods
Materials
Four wound dressings with acknowledged hemostyptic
properties were included in the study. There were two
collagen dressings: collagen SC (SuprasorbVR C,
Lohmann & Rauscher, Germany) and collagen PC
(PuracolTM, Medline Industries Inc., USA), one dress-
ing consisted of collagen and ORC: collagen þ ORC
(PromogranTM, Systagenix Wound Management,
Germany) as well as one dressing made from ORC
(TabotampVR , Johnson & Johnson, USA). In addition,
gauze (GazinVR , Lohmann & Rauscher, Germany) was
included in the study.
Blood drawing
Blood was taken from healthy volunteers (age: >20 and
<50 years) without stasis, very carefully by venipunc-
ture with “butterflies” (0.9 mm) and collected directly
under sterile conditions in pre-anticoagulated contain-
ers. The quality of the blood used for these experiments
is of utmost importance. Therefore, the following
exclusion criteria had to be strictly fulfilled: smoking,
intake of drugs in the last two weeks (especially hemo-
stasis affecting agents such as acetylsalicylic acid, oral
contraceptives, non-steroidal antiphlogistics, etc). For
anticoagulation either 3 or 1.5 IU/mL sodium heparin
was used (Ratiopharm GmbH, Germany). These spe-
cific units were chosen to simulate the different possible
applications of the wound dressings. Gauze and ORC
are used in cardiac surgery, where a high anticoagula-
tion of 3 IU/mL usually applies. The other wound
dressings are rather used on patients with low or no
Wiegand et al.
anticoagulation. Here, 1.5 IU/mL heparin was chosen
as a low anticoagulation is needed for in vitro experi-
ments. Blood drawing was specifically approved by the
Ethics Unit of the University of Tuebingen, Germany
(project approval number 270/2010BO1). Blood
donors gave their written informed consent prior to
the voluntary blood donation. Blood anticoagulated
with 0.11 M sodium citrate was used for the detection
of blood clot formation, hemolytic effects, and PMN
elastase release as recommended for the in vitro study
of biomaterials.14
Determination of blood clot formation
Thrombus formation on the dressing samples after
blood contact was inspected visually. Platelet adhesion
and aggregation, cell adhesion, and fibrinogen adsorp-
tion on the test specimen were evaluated by SEM
(Cambridge instruments, UK). The number of platelets
was determined by blood cell counting using the Cell
Counter Micros 60 (ABX Hematology, France).
Dressing samples were incubated with blood at 37 C
for 10, 20, 30, and up to 60 min in polypropylene tubes
(BD Biosciences, Germany) under static conditions. An
empty tube was used as negative control. Blood clot
formation was detected by photometric measurement
of free hemoglobin, which can be liberated from eryth-
rocytes not captured in the blood clot by osmotic lysis
in a hypotonic solution (aqua dest). The BCI was
calculated according to equation (1) from the optical
density (OD) measurement at 540 nm (BMG
Labtech, Germany).
BCI ¼ 100  OD540nm ½activated sample
(1)
=OD540nm ½non  activated control
Assessment of coagulation and platelets activation
Measurement of the TAT after contact to the dressing
samples was performed by ELISA (Siemens Healthcare
Diagnostic Products GmbH, Germany) according to
the manufacturer’s instructions. Plasmatic thrombin
generation was measured using the TechnothrombinVR
Thrombin Generation Assay (Haemochrom
Diagnostica GmbH, Germany). The assay is based on
the cleavage of the fluorogenic thrombin-specific
substrate ZGGR-7-amido-4-methylcoumarin (ZGGR-
AMC) and uses normal pooled human citrated plasma
(Haemochrom Diagnostica GmbH, Germany). The
reconstituted normal pooled plasma was incubated
with test specimens, and kinetic measurement was
carried out at 360/460 nm (ex/em) using the
SPECTROstar Omega plate reader (BMG Labtech,
Ortenberg, Germany) directly after the addition of
3
1 mM ZGGR-AMC at 37 C for 2 h. Thrombin con-
centrations in the samples were calculated on the basis
of a thrombin-standard-curve (Thrombin Calibrator,
Haemochrom Diagnostica GmbH, Germany). Peak
thrombin, lag-time, and time-to-peak were determined.
For evaluation of platelet activation by the dressing
samples, the concentration of b-TG was measured by
ELISA (Diagnostica Stago, France) according to the
manufacturer’s instructions.
Evaluation of hemocompatibility
For evaluation of hemolytic effects, erythrocytes were
isolated and directly incubated with dressing samples
for 1 h at 37 C on a shaker. For complete lysis (positive
control), erythrocytes were lysed with aqua dest, and
erythrocytes incubated in PBS alone served as negative
control. Free hemoglobin was measured as OD at
540 nm using the SPECTROstar Omega (BMG
Labtech), and the hemolysis ratio (HR) was calculated
in (%) according to equation (2)
 
OD540nm ½sample
100 
–OD540nm ½negative control
HR ¼   (2)
OD540nm ½positive control
–OD540nm ½negative control
PMN elastase released from neutrophil granulocytes
was measured after incubation of the test specimen
using a PMN elastase ELISA (Dimeditec Diagnostics
GmbH, Germany). Furthermore, the number of white
blood cells was determined by blood cell counting using
the Cell Counter Micros 60 (ABX Hematology,
France). In addition, activation of the complement
system was investigated. Complement convertase C5a
activity after 24 h was determined using the comple-
ment convertase assay according to the manufacturer’s
instructions (HaemoScan, The Netherlands) subse-
quently to incubation of the test specimens with
normal pooled human citrated plasma (HaemoScan,
The Netherlands) for 15 min at room temperature.
Three reference materials were included in the assay:
polyethylene (Ref. I), polydimethylsiloxane (Ref. II),
and medical grade steel (Ref. III) (HaemoScan, The
Netherlands). Activity of complement convertase C5a
was specified as OD after measurement time (24 h) per
test specimen surface area (cm2) [OD/24 h/cm2]. In a
second test, SC5b-9 concentrations, which serve as a
marker for activation of the terminal complement com-
plex, were measured by ELISA (Quidel, USA) accord-
ing to the manufacturer’s instructions.
4 Journal of Biomaterials Applications 0(0)

Statistical analysis (Figure 1, middle and lower panel). Slightly fewer

Experiments were performed five times for every
wound dressing for the following parameters: TAT,
b-TG, SC5b-9, BCI, platelets, white blood cells.
Experiments were performed twice for thrombin gen-
eration, HR, PMN elastase, and complement conver-
tase C5a while measurements were carried out in
quadruplicates. All values are expressed as mean  SE
(standard error). One-way analysis of variance was
carried out to determine statistical significances
(MicrosoftVR Excel 2000). Differences were considered
statistically significant at a level of p < 0.05.
Results
Thrombus formation and blood clotting
Macroscopic observations showed that all wound
dressings induce the formation of blood clots during
the 30 min incubation period. Highest blood
clotting was found for collagen SC and ORC
(Figure 1, upper panel). SEM analysis further demon-
strated the adsorption of blood cells and plasma pro-
teins by all wound dressings tested. Although
SEM does not permit quantification of adhered plate-
lets, it is possible to assess material surfaces after plate-
let adhesion visually and obtain insight into platelet
aggregation. Test specimens from collagen PC,
collagen þ ORC, and ORC were completely covered
with a thick layer of blood cells and fibrin fibers
cells were found on collagen SC, where adhesion
appeared to emerge in clusters, and gauze, which exhib-
ited less development of fibrin fibers. However, it was
also observed that cell adhesion did not occur homo-
genously on the substrates. While some areas featured
a dense packing, other parts were almost completely
free of cells.
The contact of blood with foreign material surfaces
leads to the activation and alteration of platelets with
consecutive loss of platelet functionality. Platelet
counts were very similar in the test before and after
contact of blood with gauze and ORC (Figure 2(a)).
This reflects a very low sticking of the platelets to the
surface as well as no destruction of the platelets.
However, when only 1.5 IU/mL heparin was used for
anticoagulation compared to 3 IU/mL, ORC also sig-
nificantly decreased platelet count (p < 0.05). In con-
trast, the collagen-containing samples reduced platelet
counts under both conditions in a significantly higher
degree compared to gauze (p < 0.01) as well as ORC
at 3 IU/mL (p < 0.05). No significant differences
between collagen SC, collagen PC, and collagen þ
ORC were noted.
During clot formation, the quantity of hemoglobin
entrapped by fibrin increases while at the same time the
amount of free hemoglobin decreases. This can be used
as an indicator for evaluation of the potential of
a dressing to induce clotting in the form of BCI.
It is clear that as the BCI decreases, blood clotting
increases and vice versa. An increased blood clot

(a) (b) (c) (d) (e)

Figure 1. Macro- and microscopic evaluation of the wound dressings. Blood clot formation during the 30 min incubation period with
whole blood (1.5 IU/mL heparin) on cotton gauze (a), collagen SC (b), collagen PC (c), collagen þ ORC (d), and ORC (e). Macroscopic
observations showed that all hemostyptic dressings induced the formation of blood clots (upper panel). SEM analysis of the wound
dressings demonstrated the adsorption of blood cells and plasma proteins (middle panel: 500-fold magnification, lower panel:
2500-fold magnification).
Wiegand et al. 5

(a) 3 IU/mL 1.5 IU/mL

250

200

** ** * *
Platelets [x 10E3/µL]

150
***
**

100

50

0
Negative control Gauze Collagen SC Collagen PC Collagen + ORC ORC

(b) Negative control Gauze Collagen SC Collagen PC Collagen + ORC ORC

120

100

80 **
*
BCI [%]

60 **
***

40
*
**
***
20 ***

0
–10 0 10 20 30 40 50 60 70
Time [min]

Figure 2. Effect on platelet count and blood clotting. (a) Platelet count in the samples after contact to the wound dressings using 1.5
and 3 IU/mL heparin for anticoagulation showed no effect of cotton gauze. The collagen-containing samples reduced platelet counts
under both conditions. ORC significantly decreased platelet count when 1.5 IU/mL heparin was used but not under the conditions
using 3 IU/mL heparin. (b) The potential of a dressing to induce blood clotting can be measured in the form of the BCI using citrated
blood in vitro. As the BCI decreases, blood clotting increases and vice versa. Increased blood clot formation was observed for collagen
SC, collagen PC, collagen þ ORC, and gauze observed as a significant decrease of the BCI during incubation over 30 min compared to
the control. ORC lead to a sustained BCI after 60 min compared to the control. Asterisks indicate significant deviations from the
negative control (*p < 0.05; **p < 0.01; ***p < 0.001).
BCI: blood clotting index.

formation was observed for collagen SC, collagen PC, However, ORC showed a much lower potential to
collagen þ ORC, and even gauze (Figure 2(b)), leading reduce the BCI in the test and even lead to a sustained
to a significant decrease of the BCI during incubation BCI after 60 min compared to the control and the other
over 30 min compared to the control (p < 0.001). wound dressings (p < 0.01). No significant difference
6 Journal of Biomaterials Applications 0(0)
between the collagen-containing samples and gauze
was observed.
Coagulation
Blood-material contact initiates intrinsic coagulation
via factors XII, XI, and further factor X which modu-
lates prothrombin conversion. During the reaction of
thrombin formation, the prothrombin fragment F 1 þ 2
is split off. The generated thrombin is further inacti-
vated by the formation of a complex with anti-
thrombin-III creating the so-called TAT complex,
which is an excellent marker for the detection of coag-
ulation activation. Blood after contact with the
collagen þ ORC and ORC had significantly higher
TAT levels (Figure 3(a)) compared to the control as
well as the other dressings (p < 0.01). No significant
differences between collagen ORC and ORC were
noted. If 1.5 IU/mL heparin was used instead of 3
IU/mL, the collagen dressings as well as gauze also
evoked a significant increase in TAT levels (Figure 3
(a)). The effect of gauze was however significantly
lower compared to collagen SC and collagen PC
(p < 0.01), and the effect of the sole collagen dressings
was significantly less in contrast to the ORC-containing
samples (p < 0.01). Again, no significant differences
between collagen SC and collagen PC or collagen þ
ORC and ORC were observed.
Direct measurement of thrombin generation demon-
strated significant acceleration by gauze, collagen SC,
and collagen PC in vitro, which was noticeable as a
significantly shorter lag time to onset of thrombin
generation (Figure 4(a)) and also as a significantly
decreased time-to-peak of the thrombin generation
(Figure 4(b)). No significant difference between gauze
and collagen SC for reduction of lag-time and time-to-
peak was observed. In contrast, collagen PC exhibited
a significantly higher decrease of both parameters com-
pared to gauze and collagen SC (p < 0.01).
Collagen þ ORC did not affect thrombin generation
kinetics but significantly reduced the total amounts of
generated thrombin in vitro (Figure 4(c)) compared to
gauze and collagen SC (p < 0.01) and collagen PC
(p < 0.001). It was found that collagen PC led to a sig-
nificant increase in thrombin generation in the test,
resulting in higher amounts compared to the prepara-
tions with gauze and collagen SC (p < 0.05). ORC
could not be tested in the system due to interactions
of the material with the test components.
Activation of platelets occurs in four steps: (1) shape
change with the formation of pseudopodia, (2) adhe-
sion, (3) aggregation, and (4) release of platelet factors
out of the a-granules (platelet factor 4, b-TG, etc). The
concentration of b-TG in plasma corresponds with the
degree of platelet activation. A significant increase in
b-TG levels was found for all dressings when
1.5 IU/mL heparin was used for anticoagulation
(Figure 3(b)). The effect of gauze was significantly
lower compared to the other dressings (p < 0.05). No
significant differences between the collagen and/or
ORC containing samples were noted. With 3 IU/mL,
collagen SC, collagen PC, and collagen þ ORC exerted
an increased b-TG release while ORC and gauze did
not show any activation. b-TG release was significantly
higher with collagen SC compared to collagen PC
(p < 0.05) or gauze and ORC (p < 0.01). The difference
to collagen þ ORC while noticeable was not
statistically significant. The strong activation of the
platelets observed corresponded with the reduction in
platelet count under both experimental conditions
(Figure 2(a)).
Hemocompatibility
In contact with blood, the dressings can interact with
all cells present and not only induce favorable hemo-
static effects, but could also provoke adverse biological
reactions such as hemolysis, inflammatory reactions,
and complement activation. The hemolytic activity of
a material can be assessed by the degree of erythrocy-
tolysis and dissociation of hemoglobin upon contact of
the material with blood in vitro. Absorbance values
obtained after contact of whole blood with the material
(Figure S1) are therefore transformed into the hemo-
lytic ratio (HR) according to equation (2) (Figure 5(a)).
It was found that collagen SC and gauze feature HRs
of 0.7 and 3.3, respectively. The lower HR of the col-
lagen SC indicates its better hemocompatibility com-
pared to gauze. Yet, both meet the requirements for
biomaterials with hemolysis of less than 5%.15,16 In
contrast, collagen þ ORC, ORC, and collagen PC
exhibited slightly raised HRs of 5.8, 6.5, and 8.1 indi-
cating a slight hemolytic effect. Differences between
collagen PC, collagen þ ORC, and ORC were statisti-
cally not significant.
PMN elastase is released from neutrophil granulo-
cytes during immune response and inflammatory
reactions. In the tests, gauze provoked a significant
release of PMN elastase similar to the positive control
(Figure 5(b)). The release of PMN elastase observed
was also significantly higher compared to the other
dressings (p < 0.001). None of the other materials
affected PMN elastase release. Slight differences
between collagen SC and collagen PC were not signif-
icant. Collagen ORC evoked a slightly higher PMN
elastase release compared to ORC (p < 0.05).
Interestingly, it was observed that white blood cell
counts dropped somewhat in the case of collagen SC
and collagen PC (Figure S2). It is not clear whether this
would alter cell reactivity.
Wiegand et al. 7

(a) 3 IU/mL 1.5 IU/mL

6000
**

5000
***

4000
TAT [µg/L]

3000

** ***
2000

1000 *
**
***

0
Negative control Gauze Collagen SC Collagen PC Collagen + ORC ORC

(b) 3 IU/mL 1.5 IU/mL

4000
***
3500
**

3000 **
***
Beta-thromboglobulin [IU/mL]

** ***
***
2500

2000

1500

1000
**

500

0
Negative control Gauze Collagen SC Collagen PC Collagen + ORC ORC

Figure 3. Coagulation and platelet activation. (a) The thrombin-anti-thrombin-III (TAT) complex is an excellent marker for the
detection of coagulation activation. Collagen þ ORC and ORC significantly increased TAT blood levels compared to the control as
well as the other dressings under conditions of anticoagulation with 3 IU/mL heparin. With 1.5 IU/mL heparin for anticoagulation
collagen SC and collagen PC as well as gauze also evoked an amplification of TAT levels. The dotted line indicates the level of the
negative control. (b) Platelet activation can be assessed by the corresponding concentration of b-TG in the plasma. When 1.5 IU/mL
heparin was used for anticoagulation, a significant increase in b-TG levels was found for all dressings with the exception of gauze. With
3 IU/mL heparin, collagen SC, collagen PC, and collagen þ ORC exerted an increased b-TG release while ORC and gauze did not
show any activation. The dotted line indicates the level of the negative control. Asterisks indicate significant deviations from the
negative control (*p < 0.05; **p < 0.01; ***p < 0.001).
b-TG: b-thromboglobulin.
8 Journal of Biomaterials Applications 0(0)

(a) (b)
30 35

30
* **
25
**
*** 25 ***

Time-to-peak [min]
20
Lag-time [min]

***
20
15
15
10
10

5 5
n.d. n.d.
0 0
Negative Gauze Collagen Collagen Collagen + ORC Negative Gauze Collagen Collagen Collagen + ORC
control SC PC ORC control SC PC ORC

(c) 400
***
350

300 **
Thrombin [nM]

250

200

150

100

50
n.d.
0
Negative Gauze Collagen Collagen Collagen + ORC
control SC PC ORC

Figure 4. Thrombin generation test. Assessment of coagulation by measurement of lag-time to onset of thrombin generation (a),
time-to-peak of the thrombin generation (b), and total amounts thrombin (c) using the thrombin generation test with pooled human
citrated plasma. Gauze, collagen SC, and collagen PC demonstrated accelerated formation of plasmatic thrombin noticeable as a
significantly shorter lag-time to onset of thrombin generation (a) and also significantly decreased time-to-peak of the thrombin
generation (b). Collagen þ ORC did not affect thrombin generation kinetics but reduced the total amounts of generated thrombin in
vitro (c). ORC could not be tested in the system due to interactions of the material with the test components (n.d., not determined).
Asterisks indicate significant deviations from the negative control (*p < 0.05; **p < 0.01; ***p < 0.001).

Complement activation during blood-material con-
tact takes place as a defense reaction against the sup-
posed pathological invader. For the analysis of a
potential activation of the complement system by the
dressing materials, activation of complement conver-
tase C5a was measured. Three reference materials,
polyethylene (Ref. I), polydimethylsiloxane (Ref. II),
and medical grade steel (Ref. III), were included in
the test to assess the effects of the dressings. Collagen
SC did not evoke a noticeable activation of comple-
ment convertase C5a while all other dressings had a
slight to medium effect in the test (Figure 5(c)).
Furthermore, the activation of the complement path-
way via complement factor C3a and the final end prod-
uct of the complement pathway sC5b-9, also called
MAC (membrane attack complex), may contribute to
an inflammatory reaction frequently seen as a compli-
cation of biomaterial application. In contrast to the
complement convertase assay, SC5b-9 levels reflected
significant complement activation by gauze and ORC
as well as collagen SC (Figure 5(d)). SC5b-9 levels
evoked by gauze were significantly higher compared
to the other dressings tested (p < 0.01). In contrast, in
preparations with collagen SC and ORC, SC5b-9 levels
were significantly lower compared to gauze (p < 0.01).
Collagen þ ORC and collagen PC slightly increased
SC5b-9, but differences were statistically not
significant.
Discussion
Primary function of hemostats is the prompt instiga-
tion of blood clot formation through platelet adhesion,
platelet activation, and coagulation. There have only
been few reports on comparative studies performed in
a controlled in vitro environment with human blood
Wiegand et al. 9

(a) (b)
120 2500

100
2000

PMN elastase [ng/mL]

Hemolysis ratio [%]

80
1500

60

1000
40

500
20

0 0
Negative Gauze Collagen Collagen Collagen + ORC Positive Negative Gauze Collagen Collagen Collagen + ORC Positive
control SC PC ORC control control SC PC ORC control

(c) (d) 3 IU/mL 1.5 IU/mL

2.0 2000 **
1.8 1800
C5a convertase [OD450nm/24h/cm2]

1.6 1600
1.4 1400
1.2 SC5b-9 [µg/L] 1200 ***
1.0 1000

0.8 800
***
***
0.6 600 * ** ***
** *
0.4 400

0.2 200

0 0
Ref. I Ref. II Ref. III Gauze Collagen Collagen Collagen + ORC Negative Gauze Collagen Collagen Collagen + ORC
SC PC ORC control SC PC ORC

Figure 5. Hemolysis, inflammatory reactions, and complement activation. (a) Hemolytic activity of a material is assessed by the
degree of erythrocytolysis and dissociation of hemoglobin on contact of the material with citrated blood in vitro. Absorbance values
obtained after contact of whole blood with the material are therefore transformed into the HR according to equation (2). Collagen
SC and gauze featured HR < 5% while collagen þ ORC, ORC, and collagen PC exhibited slightly raised HR > 5%. The dotted line
indicates the 5% level. (b) PMN elastase is released from neutrophil granulocytes during immune response and inflammatory reactions.
The test was carried out by incubation of the wound dressings with citrated blood in vitro. Gauze provoked a significant release of
PMN elastase similar to the positive control while none of the other materials affected PMN elastase release in vitro. The dotted line
indicates the level of the negative control. (c) Complement activation during blood-material contact was measured as activation of
complement convertase C5a. The reference materials polyethylene (Ref. I), polydimethylsiloxane (Ref. II), and medical grade steel
(Ref. III) were included in the test to assess the effects of the dressings. All dressings with the exception of collagen SC had a slight to
medium effect in the test. Collagen SC did not evoke a noticeable activation of complement convertase C5a. The dotted lines indicate
the cut-offs for inactive/low (0.2), medium (>0.2 and 0.6), and high (>0.6) reactivity classified according to assay protocol.
(d) Activation of the complement pathway was evaluated via the complement factor C3a and the final end product of the complement
pathway SC5b-9. SC5b-9 levels reflected significant complement activation by gauze and ORC as well as collagen SC. Collagen þ ORC
and collagen PC only slightly increased SC5b-9. The dotted line indicates the level of the negative control. Asterisks indicate significant
deviations from the negative control (*p < 0.05; **p < 0.01; ***p < 0.001).
HR: hemolytic ratio.

and plasma to assess the basic hemostatic properties of wound dressings on blood clotting. Platelet adhesion
commercially available hemostatic dressings.1,6,17–20 plays an important role during hemostasis. The extent
Historically, reports in this field have focused on of platelet adhesion to materials is therefore often used
animal trials using specific organ lacerations, arterial as an index for thrombogenicity. In our study, several
puncture, and skin injury models to evaluate hemostat- clotting and activation tests were used in parallel to
ic efficacy.21–26 Such animal models are still widely assess hemostyptic properties alongside with unwanted
used.20,27,28 Zhao et al.20 demonstrated good compara- activation (Table 1). As the application of the used
bility between results obtained in animal models and materials range from simple topical wound dressing
those from in vitro tests. Hence, it seems reasonable to of larger and deeper wounds (collagen-based dressings)
use an array of in vitro tests to assess the influence of to those additionally used during surgery (ORC and
10
gauze), two anticoagulation regimes to reflect these
applications have been used: 1.5 IU/ml for wound
dressings and 3 IU/ml for materials also used during
surgery. The latter concentration actually applies in
vivo during cardiac surgery using the heart–
lung machine, where an activated clotting time of
300–480 s is adjusted. Anticoagulation of blood sam-
ples is necessary to avoid spontaneous coagulation pro-
cesses in vitro. Frequently used anticoagulants are
heparin for whole blood studies and sodium citrate
for studies that focus on platelet–biomaterial interac-
tions in vitro.14 Due to the fact that heparin is routinely
applied as systemic anticoagulant in therapeutic
approaches, many laboratories apply it for whole
blood studies with biomaterials. Yet, no common stan-
dard is established for heparin resulting in the use of
different functional forms e.g. unfractionated heparin,
low molecular weight heparin, or pentasaccharide types
(e.g. Fondaparinux) and application in varying
concentrations (0.5–5 IU heparin/mL blood).14 The
1.5 IU/mL applied here is based on experience values
from hemocompatibility tests. The desired/positive
effects can be broadly grouped into clot formation
(visual inspection, SEM, BCI), platelet activation
(platelet counts, b-TG), and coagulation activation
(TAT, thrombin generation). Visual inspection of the
wound dressings after blood contact proved the forma-
tion of blood clots. SEM analysis further demonstrated
the adsorption of blood cells and plasma proteins by all
wound dressings. Although the highest blood clotting
was found for collagen SC and ORC after macroscopic
inspection, SEM yielded the following order of
platelet adhesion among the dressings: collagen PC ¼
collagen þ ORC ¼ ORC > collagen SC  cotton gauze.
While ORC for example was completely covered with a
thick layer of blood cells and fibrin fibers, slightly fewer
cells were found on collagen SC. Here, adhesion
appeared to emerge in clusters and not homogenously
on the substrate. Such effects on collagen substrates
have been previously reported by others,29 but it
seems unclear why these occur. Collagen in the body
is a natural coagulant and plays an important role in
hemostasis and thrombogenesis.11 Therefore, some of
the most widely used topical hemostatic agents nowa-
days are collagen preparations.4 However, it has been
noted that different collagen preparations vary in their
ability to aggregate platelets and support their adhe-
sion. For instance, fibrillar collagen has a much
higher potential for inducing platelet activation than
the same collagen type in acid-soluble form,30 which
led to the conclusion that the quaternary structure of
collagen is important in inducing platelet activation.11
A significantly increased blood clot formation was also
observed for the collagen-based sponges or collagen-
containing dressing and even gauze in the BCI.
Journal of Biomaterials Applications 0(0)
Yet, the ORC dressing revealed a sustained high BCI
after 60 min indicating a slower clotting rate.
Thrombin is the final product of the coagulation cas-
cade and the key enzyme of this system. Therefore,
thrombin generation is thought to reflect the overall
coagulating capacity, taking into account the effect of
all parameters influencing the coagulation system.31
There are also assays that measure the amounts of
thrombin formed by determining molecules that
result from thrombin formation and thrombin action,
e.g. D-dimers that indicate that fibrin has been formed,
F1 þ 2 that designate that prothrombin has been split,
and TAT that specifies that active thrombin has been
present.31 ORC led to a significant increase in TAT
levels compared to the collagen-based and collagen-
containing dressings or gauze. There was a distinct dif-
ference between conditions where 1.5 IU/mL heparin
was used for anticoagulation compared to 3 IU/mL.
As thrombin activation products do not represent over-
all coagulation capacity but are markers of ongoing
coagulation activation,31 it is not surprising that TAT
levels were considerably higher in blood anti-
coagulated with 1.5 IU/mL instead of 3 IU/mL hepa-
rin. Differences in test outcomes when 1.5 IU/mL
instead of 3 IU/mL heparin were used have also been
observed for the dressings concerning effects on platelet
counts and b-TG formation. Platelets were very strong-
ly activated by all dressings except for gauze, which
demonstrated statistically significant but only slight
platelet activation, when 1.5 IU/mL heparin was used
for anticoagulation. With 3 IU/mL heparin only, the
collagen-based/-containing dressings exerted increased
platelet activation. Although collagen is widely used
because of its hemostatic function, it is now generally
acknowledged that collagen is not a direct activator of
coagulation, via factor XII or any other route. Yet, it
does markedly accelerate contact (factor XII)-initiated
coagulation.1 This almost certainly reflects the
collagen-dependent activation of platelets playing a
central role in the activation of factor X by
VIIIa-factor IXa enzyme complex and prothrombin
by factors Xa/Va.32 In vivo comparative studies on
hemostatic efficacy have generally demonstrated a ben-
efit in the use of collagen-based agents over
ORC.6,21,22,24,26 In accordance, collagen-based and
collagen-containing dressings exhibited enhanced coag-
ulation activity versus ORC in this study with the
exception of the increase of TAT levels. Differences
between collagen dressings can be explained by colla-
gen preparation. Very early, it was discovered that (1)
the ability of collagen to induce platelet aggregation is
dependent on the arginine content, (2) the native fibril-
lary structure needs to be maintained for functionality,
(3) a regular arrangement of the collagen monomers in
a staggered, repetitive structure is essential for
Wiegand et al. 11

interaction with blood platelets, and (4) a particle size between the three types, depending on the test used.
of about three molecular lengths (9000Å) is the mini- Collagen SC induced a marked drop of the WBC
mum length required for platelet activation.33,34 As counts that was not observed with any other dressings.
expected, collagen preparations vary widely in their In this context, we marked this as neutral effect as the
ability to activate platelets depending on the extraction leukocytes most likely adhered to the nascent clot.
processes used during manufacture, and on the degree Monocytes express the receptor for the thrombin sub-
of chemical crosslinking and addition of other substan- strate PAR1, and neutrophils are known to express
ces, e.g. oxidized cellulose.1 The most processed colla- tissue factor and form cell–cell contacts with plate-
gen, such as gelatin, does not activate platelets at all, lets.36 Thus, the decreased WBC counts rather under-
whereas mild extraction can generate collagen prepara- line the hemostyptic properties of collagen SC.
tions that retain fibrillary content and thus initiate Looking at the negative effects, both collagen PC and
platelet adhesion and aggregation.1 collagen ORC showed slight hemolysis and comple-
As classic absorbent dressing, cotton gauze neither ment activation, which would be considered as minor
initiates nor potentiates platelet activation or clot for- activation. Here, collagen SC showed less activation of
mation.1 In accordance, gauze did not increase throm- complement and hemolysis, overall qualifying collagen
bin concentrations and only slightly affected TAT SC in this study as best hemostyptic material with
amounts and b-TG levels in vitro, although a signifi- lowest adverse reactions.
cant decrease in lag-time of thrombin generation and Comparing the wound dressings that are also used
time-to-peak was found accompanied by a reduction of during surgery, ORC undoubtedly showed stronger
hemostyptic effects than gauze, permitting its use as
the BCI compared to the control over timer. In addi-
wound dressing and in surgical procedures where
tion to the minimal thrombogenic effects, the inherent-
blood clotting is desired. Gauze on the other hand
ly adhesive nature of cotton fibers to the wound bed
does not initiate coagulation activation and clotting
and their highly absorptive qualities make cotton gauze
to a large extent. Therefore, this material is better
pads less than ideal dressings even if they have a long
used during procedures where no clotting is necessary.
standing in wound management.35 The demand for
However, the gauze dressing tested also featured the
hemostyptica is to activate coagulation and thrombus
strongest negative effects of all examined materials. It
formation for blood clotting without any adverse would be of interest to test cotton gauze pads of vari-
effects such as induction of hemolysis, instigation of ous sources to assess how different raw preparations of
inflammatory reactions, or initiation of the innate com- cotton might affect hemostatic and hemolytic proper-
plement system. In our experiments, all collagen-based ties of the final dressing pad. The processing of raw
wound dressings clearly showed desired hemostyptic cotton consists of three successive steps: desizing and
properties (Table 1), with only slight differences scouring as well as bleaching.37 It means that non-
cellulosic components such as fats, waxes, pectines,
Table 1. Summary of effects diverse wound dressings exert on and proteins are removed by alkaline treatment at
blood parameters.
high temperatures and subsequently hydrogen peroxide
Collagen Collagen Collagen is applied to destroy the residual pigments in the fibers
Effects Gauze ORC SC PC ORC rendering the natural gray of cotton to the white color
Desired or positive common for hospital grade gauze.37 As the chemical
Visual clotting – þþþ þþþ þ þþ processes only vary slightly between commercial prep-
SEM þ þþþ þþ þþþ þþþ arations, similar effects on fiber structure and quality
BCI þþ – þ(þ) þþþ þþþ can be expected leading to comparable efficacies in
Platelet counts – þþ(þ) þþþ þþ þþ
dressing performance.
b-TG (þ) (þþþ) þþ þþ þþ
Thrombin þ n.d. þþ þþþ –
TAT þ þþþ þþ þþ þþþ Conclusions
Neutral
WBC counts (–) (–) –– (–) (–) Hemostatic properties of materials can be assessed in
Negative vitro by testing their effects on activation of platelets
HR þ þ (þ) þ(þ) þ
PMN elastase þþ – – – –
and induction of coagulation. To reliably predict the
C5a þ þ – þ þ effects of hemostatic wound dressings in vitro, tests
SC5b-9 þþ þ þ þ þ have to be chosen with regard to sensitivity as well as
physiological situation and intended use of the materi-
Desired or positive ¼ hemostyptic effects, neutral ¼ effects likely related
to clotting, negative ¼ unwanted activation effects.
al. For instance, dressings intended for clotting activa-
–, no effect; – –, reduction; þ, low increase; þþ, medium increase; þþþ, tion in cardiac surgery need to be tested under
high increase; n.d., not determined. conditions with high anticoagulation and very sensitive
12
tests because of the potential risk to the patient.
Moreover, hemocompatibility needs to be carefully
considered as there is a link between complement acti-
vation and inflammatory properties. Thus, it is very
important to choose a variety of tests meshing desired
and undesired effects of the intended use. For hemos-
typtica, the activation of coagulation and thrombus
formation without triggering any adverse events is cru-
cial. Our study therefore shows that collagen-based
dressings are ideal hemostyptic wound dressings with
a very good benefit/safety ratio. ORC and collagen
ORC do not show any improved or additive effects.
Gauze, in contrast to the common practice, is far
less optimal as wound dressing or during surgery.
Moreover, our experiments reveal the necessity of com-
bining a range of intertwining in vitro tests to predict
the clotting or inflammatory behavior of materials.
However, there currently is no direct transfer of the
in vitro tests to the in vivo situation although hemos-
typtic properties of wound dressings are likely quite
similar in vitro and in vivo due to the topical applica-
tion and contact with exudate and blood only.
Acknowledgements
The authors would like to thank Doreen Winter for excellent
technical assistance.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of
interest with respect to the research, authorship, and/or pub-
lication of this article: Author MA is an employee of
Lohmann & Rauscher GmbH.
Funding
The author(s) disclosed receipt of the following financial sup-
port for the research, authorship, and/or publication of this
article: Lohmann & Rauscher GmbH Germany.
ORCID iD
Cornelia Wiegand http://orcid.org/0000-0001-7802-2785
Supplemental Material
Supplemental material for this article is available online.
ORCID iD
Cornelia Wiegand http://orcid.org/0000-0001-7802-2785
References
1. Jesty J, Wieland M and Niemiec J. Assessment in vitro
of the active hemostatic properties of wound dressings.
J Biomed Mater Res Part B Appl Biomater 2009;
89B: 536–542.
Journal of Biomaterials Applications 0(0)
2. Martin P. Wound healing – aiming for perfect skin regen-
eration. Science 1997; 276: 75–81.
3. Lewis KM, Spazierer D, Slezak P, et al. Swelling, sealing,
and hemostatic ability of a novel biomaterial: a polyeth-
ylene glycol-coated collagen pad. J Biomater Appl 2014;
29: 780–788.
4. Broekema FI, van Oeveren W, Zuidema J, et al. In vitro
analysis of polyurethane foam as a topical hemostatic
agent. J Mater Sci Mater Med 2011; 22: 1081–1086.
5. Tomizawa Y. Clinical benefits and risk analysis of topical
hemostats: a review. J Artif Organs 2005; 8: 137–142.
6. Wagner WR, Pachence JM, Ristich J, et al. Comparative
in vitro analysis of topical hemostatic agents. J Surg Res
1996; 66: 100–108.
7. Pathak CP, Sawhney AS, Quinn CP, et al.
Polyimidepolyethylene glycol block copolymers: synthe-
sis, characterization and initial evaluation as biomaterial.
J Biomater Sci Polym Ed 1994; 6: 313–323.
8. Krajewski S, Hierlemann T, Neumann B, et al.
Hypercoagulant abdominal swabs in cardiac surgery:
potential problems and background. Thorac Cardiovasc
Surg 2016; 64: 589–595.
9. Krajewski S, Nathan T, Neumann B, et al. Simple clot-
ting test to detect procoagulant abdominal swabs.
J Mater Sci Mater Med 2015; 26: 106.
10. Schonauer C, Tessitore E, Barbagallo G, et al. The use of
local agents: bone wax, gelatin, collagen, oxidized cellu-
lose. Eur Spine J 2004; 13: S89–S96.
11. Kawamoto Y and Kaibara M. Procoagulant activity
of collagen. Effect of differences in type and structure
of collagen. Biochim Biophys Acta 1990; 1035: 361–368.
12. Friess W. Collagen – biomaterial for drug delivery. Eur J
Pharm Biopharm 1998; 45: 113–136.
13. Pusateri AE, Holcomb JB, Kheirabadi BS, et al. Making
sense of the preclinical literature on advanced hemostatic
products. J Trauma 2006; 60: 674–682.
14. Braune S, Grunze M, Straub A, et al. Are there sufficient
standards for the in vitro hemocompatibility testing of
biomaterials? Biointerphase 2013; 8: 33.
15. Chen Y, Zhang Y, Wang F, et al. Preparation of porous
carboxymethyl chitosan grafted poly (acrylic acid) super-
absorbent by solvent precipitation and its application as
a hemostatic wound dressing. Mater Sci Eng C 2016;
63: 18–29.
16. Zhen Z, Liu X, Xi TF, et al. Hemolysis and cytotoxicity
mechanisms of biodegradable magnesium and its alloys.
Mater Sci Eng C Mater Biol Appl 2015; 46: 202–206.
17. Eloy R, Baguet J, Christe G, et al. An in vitro evaluation
of the hemostatic activity of topical agents. J Biomed
Mater Res 1988; 22: 149–157.
18. Rembe JD, B€ ohm JK, Fromm-Dornieden C, et al.
Comparison of hemostatic dressings for superficial
wounds using a new spectrophotometric coagulation
assay. J Transl Med 2015; 13: 375.
19. Sawyer PM, Schwann G and Stanczewski B. Effects of a
new hemostatic agent on blood coagulation. Biomater
Med Dev Artif Organ 1983; 11: 135–145.
20. Zhao X, Guo B, Wu H, et al. Injectable antibacterial
conductive nanocomposite cryogels with rapid shape
Wiegand et al.
recovery for noncompressible hemorrhage and wound
healing. Nat Commun 2018; 9: 2784.
21. Zoucas E, Goransson G and Bengmark S. Comparative
evaluation of local hemostatic agents in experimental liver
trauma: a study in the rat. J Surg Res 1984; 37: 145–150.
22. Chvapil M, Owen JA and DeYoung DW. A standardized
animal model for evaluation of hemostatic effectiveness
of various materials. J Trauma 1983; 23: 1042–1047.
23. Coln D, Horton J, Ogden ME, et al. Evaluation of hemo-
static agents in experimental splenic lacerations. Am J
Surg 1983; 145: 256–259.
24. Silverstein ME, Keown K, Owen JA, et al. Collagen
fibers as a fleece hemostatic agent. J Trauma 1980;
20: 688–694.
25. Abbott WM and Austen WG. The effectiveness and
mechanism of collagen-induced topical hemostasis.
Surgery 1975; 78: 723–729.
26. Hait MR, Robb CA, Baxter CR, et al. Comparative eval-
uation of avitene microcrystalline collagen hemostat
in experimental animal wounds. Am J Surg 1973;
125: 284–287.
27. Qu J, Zhao X, Liang T, et al. Antibacterial adhesive
injectable hydrogels with rapid self-healing, extensibility
and compressibility as wound dressing for joints skin
wound healing. Biomaterials 2018; 183: 185–199.
28. Zhao X, Wu H, Guo B, et al. Antibacterial anti-oxidant
electroactive injectable hydrogel as self-healing wound
dressing with hemostasis and adhesiveness for cutaneous
wound healing. Biomaterials 2017; 122: 34–47.
13
29. Boccafoschi F, Habermehl J, Vesentini S, et al. Biological
performances of collagen-based scaffolds for vascular
tissue engineering. Biomaterials 2005; 26: 7410–7417.
30. Savage B, Ginsberg MH and Ruggeri ZM. Influence of
fibrillar collagen structure on the mechanisms of platelet
thrombus formation under flow. Blood 1999; 94: 2704–2715.
31. Nair SC, Dargaud Y, Chitlur M, et al. Tests of global
haemostasis and their applications in bleeding disorders.
Haemophilia 2010; 16: 85–92.
32. Walsh PN. Platelet coagulation-protein interactions.
Semin Thromb Hemost 2004; 30: 461–471.
33. Wang CL, Miyata T, Weksler B, et al. Collagen-induced
platelet aggregation and release. I. Effects of side-chain
modifications and role of arginyl residues. Biochim
Biophys Acta 1978; 544: 555–567.
34. Wang CL, Miyata T, Weksler B, et al. Collagen-induced
platelet aggregation and release. II. Critical size and
structural requirements of collagen. Biochim Biophys
Acta 1978; 544: 568–577.
35. Rich PB, Douillet C, Buchholz V, et al. Use of the novel
hemostatic textile StasilonVR to arrest refractory retroper-
itoneal hemorrhage: a case report. J Med Case Rep 2010;
4: 20.
36. Bouchard BA and Tracy PB. The participation of leuko-
cytes in coagulant reactions. J Thromb Haemost 2003;
1: 464–469.
37. Tzanova T, Calafellb M, Guebitzc GM, et al. Bio-prep-
aration of cotton fabrics. Enzyme Microbial Technol
2001; 29: 357–362.