Acta Biomaterialia xxx (xxxx) xxx

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Acta Biomaterialia
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An in vitro model mimics the contact of biomaterials to blood

components and the reaction of surrounding soft tissue
Maren Jannasch a, Sabine Gaetzner a, Florian Groeber b, Tobias Weigel a, Heike Walles a,b, Tobias Schmitz a,
Jan Hansmann a,b,⇑
a
University Hospital Wuerzburg, Department Tissue Engineering and Regenerative Medicine (TERM), Roentgenring 11, 97070 Wuerzburg, Germany
b
Fraunhofer Institute for Silicate Research ISC, Translational Center Regenerative Therapies, 97070 Wuerzburg, Germany

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

Article history: The therapeutic efficacy of a medical product after implantation depends strongly on the host-initiated
Received 2 August 2018 fibrotic response (foreign body reaction). For novel biomaterials, it is of high relevance to understand this
Received in revised form 20 February 2019 fibrotic process. As an alternative to in vivo studies, in vitro models mimic parts of the whole foreign body
Accepted 13 March 2019
reaction. Aim of this study was to develop a wound model with key cells and matrix proteins in coculture.
Available online xxxx
This approach combined blood components such as primary macrophages in a plasma-derived fibrin
hydrogel, directly exposed to reference biomaterials (PTFE, glass, titanium). The soft tissue reaction is
Keywords:
resembled by integrating fibroblasts in a collagen or a fibrin matrix. Those two experimental setups were
Foreign body reaction
Test system
conducted to show whether a long-term in vitro culture of 13 days is feasible. The response to reference
Three-dimensional tissue biomaterials was assessed by multi-parametric analyses, comprising molecular profiling (cytokines, col-
In vitro model lagen I and ß-actin) and tissue remodeling (cell adherence, histological structure, tissue deposition).
Blood components Polytetrafluorethylene (PTFE) and titanium were tested as references to correlate the in vitro evaluation
to previous in vivo studies. Most striking, both model setups evaluated references’ fibrotic characteristics
as previously reported by in vivo studies.

Statement of Significance

We present a test platform applied for assessments on the foreign body reaction to biomaterials. This test
system consists of blood components – macrophages and plasma-derived fibrin - as well as fibroblasts
and collagen, generating a three-dimensional wound microenvironment. By this modular approach, we
achieved a suitable test for long-term studies and overcame the limited short-term stability of whole
blood tests. In contrast to previous models, macrophages’ viability is maintained during the extended cul-
ture period and excels the quality of the model. The potential to evaluate a foreign body reaction in vitro
was demonstrated with defined reference materials. This model system might be of high potential as a
screening platform to identify novel biomaterial candidates.
Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The implantation of a biomaterial into a tissue disturbs its

Despite the scientific effort to advance biomaterials towards a
low immune reactivity, all medical products induce adverse reac-
tions such as inflammation or fibrosis [1,2].
Abbreviations: IL, interleukin; 3D, three-dimensional.
⇑ Corresponding author at: Fraunhofer Institute for Silicate Research ISC, Trans-
lational Center Regenerative Therapies, Roentgenring 11, 97070 Wuerzburg,
Germany.
E-mail address: Jan.Hansmann@isc.fraunhofer.de (J. Hansmann).
homeostasis and entails as a response to the injury a bleeding. In
consequence, the biomaterial is immediately exposed to blood pro-
teins, adsorbing on its surface. The surface exposition as well as the
injury itself activates the coagulation cascade. Thereby, a first fibrin
clot forms the interface to the native collagen-based tissue matrix,
the foreign body is embedded in a matrix net, and finally the defect
site is provisionally closed. Alarm signals in response to injury (e.g.
degraded matrix proteins, intracellular proteins) or in response to
surface contact (e.g. complement factors) stimulate a directed infil-
tration of immune cells to the implant region. Adsorbed blood

https://doi.org/10.1016/j.actbio.2019.03.029
1742-7061/Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article as: M. Jannasch, S. Gaetzner, F. Groeber et al., An in vitro model mimics the contact of biomaterials to blood components and the
reaction of surrounding soft tissue, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2019.03.029
2 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx
plasma proteins (e.g. fibronectin) on the biomaterial serve as adap-
tors for cell adhesion [3,4]. Granulocytes mediate by proteases and
reactive oxygen species a structural degradation of the biomaterial
and the surrounding tissue. Within days, those short-lived granu-
locytes are replaced by entering macrophages [5]. Limited by size
and degradability, macrophages fail to phagocytose the foreign
body [6]. Nevertheless, macrophages persist directly on the sur-
face, form a first cell layer, and fuse to multi-cellular ‘‘gigantic”
cells – a central process during the isolation from the internal body
[7]. Through surface contact during the whole implant lifetime,
macrophages detect the foreign structure and translate this signal
in a secretion of soluble mediators (e.g. cytokines, proteases). This
inflammatory milieu stimulates fibroblasts in the proximity of the
implant to chemotaxis, proliferation, and collagen synthesis [3].
Over weeks, the foreign body gets completely covered by cells
and matrix proteins. This fibrous isolation maintains the organ-
isms’ safety. However, it hinders in turn a direct integration of an
implant in the ‘‘functional” tissue.
To ensure an intended therapeutic efficacy, it is a need to under-
stand the fibrotic process that is induced by an implant and appro-
priate test procedures are absolutely essential. The gold standard
is to assess the local effect after implantation in animals [8]. Pro-
ceeding weeks after implantation, the tissue integration is analyzed
by cell infiltrates or deposited fibrous tissue [8,9]. However, physio-
logical differences between organisms cause a low correlation
between pre-clinical and clinical studies [10]. In vitro tests based
on human cells and tissue bear the potential to mimic the human
system closely [11]. Addressing a conscious and carefully reasoned
application of animals, reliable in vitro tests might also reduce the
burden on animals in science [12]. However, international standards
for tests (ISO) do not cover this area of medical device evaluation
adequately. Currently, ISO 10993-4 addresses the in vitro and
in vivo evaluation of medical devices directly and indirectly in con-
tact with blood. This analysis is aimed at devices that contact the
bloodstream or vasculature and focuses on thrombosis specifically
[13]. ISO10993-4 describes no test for implants in soft tissue that
transiently contact blood during implantation. ISO 10993-6 whereas
covers the evaluation of local effects following implantation of med-
ical devices, such as the foreign body reaction. Importantly, all these
recommendations focus on the use of appropriate animal models
[8]. Currently, there is no standardly accepted in vitro model system
to evaluate the foreign body response. To assess the fibrotic
response, previous in vitro tests apply as scaffolds, harboring respon-
sive cells, single matrix components such as collagen, gelatin and
alginate (see Table 1) [14–16]. None of these existing in vitro models
incorporates both the transient exposure of the biomaterial to blood
and the 3D fibrin matrix that is immediately formed around the
implant. As this occurs for all implants due to the surgical procedure,
current in vitro test systems might be improved by mimicking the
wound microenvironment after implantation.
A suitable wound model takes steps to incorporate relevant
aspects of the implant scenario: transient exposure to blood and
fibrin matrix as well as the integration of key cell types. Neverthe-
less, the limited stability over time substantiates the existing chal-
lenge to integrate and to maintain whole blood in a test procedure.
This limitation strengthened our previous approaches to integrate
blood components rather than whole blood in an in vitro test set-
ting [17,18]. In a human in vitro test, we compared various clini-
cally relevant scenarios such as lipopolysaccharide contamination
or the presence of IL-4 on the cytokine secretion profiles of macro-
phages in contact with biomaterials. Those conditions were evalu-
ated by the response pattern to reference biomaterials. Therefore,
cytokine profiles were incorporated in a statistical model. The
resulting patterns were correlated to in vivo studies and the surface
treatment with human blood plasma had been demonstrated as a
convincing test condition [17]. This simple model is cost efficient
Please cite this article as: M. Jannasch, S. Gaetzner, F. Groeber et al., An in vitro model mimics the contact of biomaterials to blood components and the
reaction of surrounding soft tissue, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2019.03.029
and easy for other labs to adopt. However, the assay facilitates only
to analyze the acute response within 48 h. Especially for medical
products with active parts such as digestive compounds, sensors
or pharmaceutical ingredients, a test is required, which character-
izes a specific function over a longer time period and finally allows
a continuous observation.
In vitro, macrophages are able to cover a biomaterial surface
within 48 h [17]. In contrast, after implantation of a biomaterial,
the signal transduction from macrophages to fibroblast and their
translation in a matrix remodeling is a cumulative long-term
effect. This reasoned a further study, where we exposed macro-
phages to reference biomaterials and collected the conditioned
medium at 48 h. In response to this conditioned medium that con-
tained released factors from the macrophages, we characterized
after 14 days the tissue remodeling by fibroblasts integrated in a
collagen hydrogel. By this decoupled test setup, a translation of
the macrophage-mediated signal into a biomaterial-dependent
matrix deposition and contraction was identified [18]. However,
our established decoupled procedure catches the biomaterial’s
effect exclusively at one specific time point and mitigates the
direct continuous cross-talk between macrophages and fibroblasts.
Thus, as a next step, we aimed to mimic a continuous long-term
interface between the biomaterial and the ‘‘sensory” macrophages,
as well as a continuous ‘‘live” diffusion of soluble mediators to the
‘‘responsive” fibroblasts.
Therefore, a 3D system was developed, which mimics the
wound niche and the fibrotic tissue formed in the proximity of
an implant (see Fig. 1): where bleeding transiently occurs, fibrin
is formed as a first matrix embedding the implant, and macro-
phages deposit on the biomaterial surface. In response to
macrophage-mediated signals, fibroblasts are stimulated to from
a fibrous capsule. To show the feasibility of a stable long-term cul-
ture, we compared two matrix environments: (I) comprising
macrophages and fibroblasts in fibrin hydrogels as well as (II) com-
bining fibrin-embedded macrophages with fibroblasts in a collagen
hydrogel. After the continuous culture for 13 days, a set of read-
outs, characterizing inflammation, proliferation and matrix deposi-
tion, was analyzed. Taking all parameters together, a principal
component analysis allowed a screening for reaction patterns
between experimental groups. This screening tool was applied to
show the capacity of both models to discriminate between tita-
nium and PTFE. A comparison with previous in vivo studies allowed
a correlation to the in vitro studies: titanium exhibits in vivo a low
fibrous tendency, whereas PTFE induces a moderate fibrosis [19–
22] (see discussion and summary Table 3 for more details).
With our development, we identified two systems, which main-
tain the viability of integrated macrophages and fibroblasts in a
long-term culture. Finally, we established a test procedure from
bench to data analysis that facilitates an evaluation of biomaterials’
fibrotic characteristics and verified the test principle by the com-
parison of titanium and PTFE in the model system. Those results
matched to expected fibrotic tendencies as observed in vivo. Most
outstanding, both models came to a corresponding outcome and
thereby strengthen our effort to develop and to optimize in vitro
systems for biomaterial screenings.
2. Materials and methods
Technically, monocytes were isolated from blood and inte-
grated in a plasma-derived fibrin clot directly exposed to the test
surface (see Fig. 1). As a carrier, a cell culture insert was placed
on top of this surface-exposed fibrin clot. In this cell culture insert,
fibroblasts were seeded either in a plasma-derived fibrin or a col-
lagen hydrogel. Those approaches were considered to mimic the
migration zone of fibroblasts in a wound niche (fibrin model) or
 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx 3

Table 1
Summary of previous published in vitro models to assess effects of biomaterials.

Author (Year) Matrix Cells Design Endpoint Readout

[Ref]
Zhu [64] collagen I human monocytes from blood; fetal 3D coculture day 5, 10, 15 and 21 gel contraction
fibroblast cell line HFL-1 from lung
Zeng [14] marine gelatine murine peritoneal macrophages, dermal 3D coculture 3, 7, 10 and 14 days proliferation, migration,
and alginate fibroblasts distribution, cytokines
Holt [65] non murine macrophage cell line RAW 264.7 2D monocultures, 21 days cytokines, cell adherence and
and primary bone marrow-derived conditioned cultures, morphology
macrophages, fibroblast-like cell line direct and indirect
NIH 3 T3 cocultures
Chai [66] acellular human primary oral keratinocytes and 3D coculture 11–13 days histology
human- fibroblasts
cadaveric dermis
Sanchez [67] agarose murine monoculture of macrophages 3D monoculture 7–14 days viability, histology
from bone marrow, pre-differentiated
with M CSF
Pan [68] non murine RAW 264.7 macrophages, M. 2D coculture and days 3, 7 and 21 cell attachment, morphology,
DUNNI dermal fibroblasts monculture distribution, viability, oxidative
burst, nitric oxide, cytokines
Grotenhuis [69,70] non human primary blood-derived 2D monoculture 3 days gene expression, cytokines
monocytes
Parks [16] collagen I human LPS-activated monocyte cell line 3D coculture 4 h, 24 h, 96 h, cytokines, morphology
U937, dermal fibroblasts 7 days and 10 days
Damanik [71] agarose rat alveolar macrophage cell line 3D monoculture on day 1 and day 3 for cell attachment, morphology,
NR8383, neonatal dermal fibroblasts agarose mould and 2D monoculture; day 1, proliferation, cytokines, protein
conditioned culture 4 and 7 for coculture expression of collagen and
elastin
Novak [72] fibrin hydrogels murine RAW 264.7 macrophages or 3 T3 3D monoculture 24 h cell viability, sensor response
fabricated from fibroblast/pre-adipocyte cell line
fibrinogen
solution
Jannasch [18] collagen human primary macrophages and 3D conditioned culture 14 days gel contraction, histology
hydrogels dermal fibroblasts of fibroblasts
Jannasch [17] plasma-derived human primary macrophages 3D culture of 2 days cytokines, cell viability,
fibrin hydrogels macrophages histology

the interface to the native soft tissue (collagen model). A continu-
ous exchange of soluble mediators was maintained through a
membrane in the applied cell culture insert, connecting both parts.
In addition to tests on titanium and polytetrafluorethylene (PTFE),
the influence of single components on the system should be shown
by tests on glass: here for monocultures with macrophages or
fibroblasts or matrix models without cells were investigated. After
13 days in culture, molecular and histological analyses were
conducted.
using a titanium target (120 mm diameter, 10 mm height) with a
target-to-substrate distance of 100 mm, titanium films were in-
house deposited on glass bowls (34 mm diameter, Brandt,
Wertheim, Germany). The external manufacturer Rhenotherm
GmbH (Kempen, Germany) applied on glass bowls a PTFE layer
(Rhenolase MK I-grau). Prior testing, test samples were sterilized
by ultrasonic treatment with deionized water for 30 min, subse-
quent incubation in 70% ethanol for 15 min and autoclave
sterilization.

2.1. Ethical clearance statement
Granted by the ethics committee of the Julius-Maximilians-
University Wuerzburg (vote 182/10), five human blood samples
were obtained under informed signed consent according to the
ethical approval (University Hospital Wuerzburg, Germany). Fro-
zen human blood plasma was purchased from the Bavarian Red
Cross blood donation service (Blutspendedienst des Bayerischen
Roten Kreuzes, München, Germany). As defined in the Declaration
of Helsinki, the experiments with blood products were conducted
in compliance with the rules for investigation on human subjects.
2.2. Pooled plasma
Frozen citrate-buffered human plasma (n = 10) was thawed for
2.5 h at 37 °C. For each donor, equal volumes of the obtained
human plasma were pooled (n = 10) and stored at - 80 °C. For fur-
ther use the frozen plasma was thawed as previously described.
2.3. Preparation of test materials
Titanium, glass, and PTFE samples were prepared as previously
applied [17,18]. Briefly, by radio frequency magnetron sputtering
2.4. Isolation of human monocytes and integration in surface-exposed
fibrin hydrogels
Mononuclear cells were isolated from leukocyte concentrate by
ficoll gradient centrifugation (GE Healthcare, Freiburg, Germany).
To purify monocytes from the generated cell fraction, a negative
magnetic cell separation selecting T cells, NK cells, B cells, dendritic
cells, and basophils was performed (Miltenyi Biotec, Bergisch Glad-
bach, Germany). Following the isolation, the obtained monocytes
(1 * 106 cells per model) were directly integrated in a plasma-
derived fibrin gel. As previously described by others [23,24],
800 ml plasma was equally distributed on materials’ surface
(7 cm2) and 100 ml cell suspension (1 * 107 monocytes per ml
coculture medium) were added (see Figs. S1 and S2 for the per-
formed medium tests with fibroblasts and monocytes). This cocul-
ture medium consisted of RPMI (Gibco) supplemented with 10%
fetal calf serum (Gibco), 1 mM sodium pyruvate (Gibco; supports
energy metabolism via citric acid cycle), 100 mM ascorbic acid
phosphate (Sigma Aldrich, Muenchen, Germany; supports as a
cofactor collagen synthesis) and 160 mg per ml t-AMCA (Pfizer,
Muenster, Germany; see Fig. S4 for information on its intended
use as fibrin stabilizer). To construct cell-free fibrin hydrogels,
100 ml coculture medium was mixed with the surface-applied

Please cite this article as: M. Jannasch, S. Gaetzner, F. Groeber et al., An in vitro model mimics the contact of biomaterials to blood components and the
reaction of surrounding soft tissue, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2019.03.029
4 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx

Fig. 1. Wound models mimic the three-dimensional (3D) interface between the material surface, the wound niche and the native soft tissue. (A) The developed coculture
system combined cell and matrix components, which resemble the wound microenvironment after implantation of a biomaterial. (B) A plasma-derived fibrin matrix
embedding monocytes was applied directly on biomaterials’ surface. Supported by a standing cell culture system, fibroblasts were integrated in a hydrogel consisting of
either fibrin or collagen. (C) The wound models were cultured under M-CSF stimulation for 6 days and grown subsequently to day 13. (D) To assess the effect of different
materials i. collagen models and ii. fibrin models were applied on glass, PTFE and titanium; iii. monocultures with fibroblasts or with macrophages as well as matrix models
without cells were constructed to investigate the influence of single components on the complex system.

plasma. Finally, the anti-coagulation of the stabilized plasma was
reversed by adding 100 ml thrombin (20 enzymatic units per ml;
Sigma Aldrich, Muenchen, Germany) in 150 mM calcium chloride
solution (Sigma Aldrich, Muenchen, Germany). To support fibrin
synthesis, this mixture was incubated for 30 min at 37 °C.
2.5. Apical hydrogel with human dermal fibroblast cell line HFF-1 in a
cell culture insert
The fibroblast cell line HFF-1 (ATCC, Wesel, Germany) was
seeded in a density of 4 * 103 cells per cm2. The medium consisting
of DMEM (Gibco, Carlsbad, USA) plus 10% fetal calf serum
(41F1142K, Gibco) and 1 mM sodium pyruvate (Gibco) was
exchanged on every second to third day. In a cell culture insert
(PIHP01250, Merck, Darmstadt, Germany), integrating a permeable
polycarbonate membrane bottom with pores of 0.4 mm in diame-
ter, (I) plasma-derived fibrin hydrogels and (II) collagen hydrogels
were constructed. All cell-based models comprised 2 * 105 HFF-1
cells (passage 18) in a final volume of 500 ml.
2.5.1. (I) fibrin models
The plasma-derived fibrin hydrogel was compounded as
described in previous section. In short, 400 ml plasma was mixed
with 50 ml cell suspension (4 * 106 cells per ml coculture medium)
and 50 ml thrombin (20 enzymatic units per ml) in 150 mM cal-
cium chloride solution. To construct cell-free hydrogels, 50 ml
coculture medium was applied.
2.5.2. (II) collagen models
For the collagen models, HFF-1 fibroblasts (4 * 105 cells per ml)
were suspended in 500 ml collagen hydrogels (final concentration
of 7 mg per ml rat-tail collagen, in-house manufactured, Wuerz-
burg, Germany). In addition, cell-free collagen hydrogels were gen-
erated by mixing collagen solution in a 2:1 ratio with gel
neutralizer solution (90 mM HEPES, 0.1 mM chondroitin sulfate,
3% FCS in 2-fold-concentrated DMEM; pH8.5). Following a gelation
for 30 min at 37 °C, the cell culture inserts were positioned on the
material-exposed fibrin gel.
2.6. Replicates and culture of the models
Per primary monocyte donor (n = 5), three technical replicates
(n = 3) were constructed on glass, titanium and PTFE. Additionally,
monocultures with macrophages or fibroblast as well as matrix
models without cells were constructed on glass (n = 3). On each
model, 2 ml coculture medium was applied and renewed on every
second to third day. To boost differentiation in the first 6 days,
macrophages were stimulated with M-CSF (40 ng per ml; Pepro-
tech, New Jersey, United States). Based on previous experience,
the models were cultured for 13 days to further analyze tissue
remodeling [18]. Reasoned on the respective procedure, replicates

Please cite this article as: M. Jannasch, S. Gaetzner, F. Groeber et al., An in vitro model mimics the contact of biomaterials to blood components and the
reaction of surrounding soft tissue, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2019.03.029
 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx
per primary cell donor (n = 5) were analyzed as follows: one repli-
cate was fixed for histology, two replicates were analyzed for wet
weight of the hydrogels and macrophages’ viability on the surface.
Finally, in the supernatant of all three replicates the cytokine pro-
file was analyzed, whereas one replicate was applied for the west-
ern blot.
2.7. Wet weight of the fibrin clot on materials’ surface and of the
connective tissue in the insert
On day 13, the medium was collected, the fibrin clot was
removed from biomaterials’ surface and the tissue was transferred
to a vial. The collected fibrin hydrogel was weighted (Kern und
Sohn, Munich, Germany). Afterwards, the apical hydrogels were
analyzed.
2.8. Macrophages’ viability on materials’ surface
Once the plasma-derived fibrin hydrogel was removed, the sur-
face was washed with phosphate buffered saline (Gibco) and the
CellTiter assay was performed according to the manufacturer’s
procedure (Promega, Mannheim, Germany). In detail, CellTiter
reagent was applied in a ratio of 78 ml per cm2 and luminescence
was measured in duplicates with a microplate Tecan Reader infi-
niteÒ M200 (Crailsheim, Germany). Per primary cell donor
(n = 5), cells’ viability was assessed on to test surfaces.
2.9. Cytokines in the supernatant
Before analysis, the collected cell culture supernatant was cen-
trifuged for 5 min at 10.000  g0. According to the manufacturer’s
protocol, an ELISA (BMS249, eBioscience) was applied to analyze
in duplicates human TGF-b1. According to manufacturer’s protocol,
IL-1ß, IL-6, IL-8, IL-10, IL-12 and TNF-a were characterized apply-
ing a human inflammatory CBA (551811, BD Biosciences, Heidel-
berg, Germany). The analysis was performed with a FACS Calibur
(BD Biosciences) and data were further processed with FCAP Array
Software 3.0 (BD Biosciences). For each primary cell donor, the
supernatant of three models was assessed.
2.10. Histochemical staining of macrophages on glass
Models were fixed in 4% paraformaldehyde (Carl Roth, Karl-
sruhe, Germany) over night at 4 °C. Histochemical staining was
performed according to a standard protocol. Macrophages’ mor-
phology on glass was stained by an anti-human ICAM-1 antibody
(AH55411, Invitrogen, Carlsbad, USA) in a 1:100 dilution and an
anti-human CD163 antibody in a 1:200 dilution (PA5-32308, Invit-
rogen, Carlsbad, USA) overnight at 4 °C. Following, the secondary
anti-mouse IgG Alexa Fluor 488 (A-21202, Invitrogen) and the sec-
ondary anti-rabbit IgG Alexa Fluor 647 (A-31573, Invitrogen) were
incubated in a 1:500 dilution for 60 min at room temperature. The
staining was mounted in Fluoromount plus Dapi (eBioscience).
Images were captured on a confocal laser-scanning microscope
(TCS SP8, Leica Microsysteme Vertrieb GmbH, Wetzlar, Germany).
Background subtraction and contrast enhancement were per-
formed on all images equally (ImageJ 1.49 m, National Institutes
of Health, Bethesda, United States).
2.11. Histochemical staining
According to the manufacturer’s protocol (Morphisto, Frankfurt,
Germany), paraffin-embedded tissue sections of 5 mm thickness
were Azan-stained. The staining was sealed with Entellan (Merck,
Darmstadt, Germany). Furthermore, tissue sections (5 mm thick-
ness) of the apical connective tissue unit were stained for Collagen
Please cite this article as: M. Jannasch, S. Gaetzner, F. Groeber et al., An in vitro model mimics the contact of biomaterials to blood components and the
reaction of surrounding soft tissue, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2019.03.029
5
III, applying a primary anti-human antibody (HPA007583, Sigma
Aldrich) in a 1:200 dilution and incubating it over night at 4 °C.
The bound primary antibodies were detected by an antibody-
mediated HRP-DAB-detection system (DCS Diagnostics, Hamburg,
Germany). Following, a Mayer’s acidic Haematoxylin (in-house
manufactured) counter staining of the cell nuclei was performed.
Images were taken on a fluorescence microscope BZ-9000 (Key-
ence, Neu-Isenburg, Germany). With the software ImageJ, the cyan
mean intensity (CMI) of Azan-stained tissue sections was analyzed
to quantify tissue remodeling. Therefore, each image was decom-
posed to CMYK-channel images and respective cyan channel image
was converted to 8-bit grey scale. Grey scale histograms allowed
the CMI-read-out. Following the CMI-analysis, the Azan-stained
images were equally contrast enhanced.
2.12. Raster electron microscopy
The tissue was fixed for 20 min in 6% glutardialdehyde, dehy-
drated in an increasing acetone series, critical point dried in an
automated system (Baltic Präparation, Niesgau, Germany) and a
1-nm-thick platin coating was applied (Leica Mikrosysteme, Wet-
zlar, Germany). The images were taken with an accelerating volt-
age of 2 kV on a raster scanning microscope Supra 25 (Zeiss,
Oberkochen, Germany).
2.13. Transmission electron microscopy
Before ultrastructural analysis, the apical hydrogel was fixed in
1% formalin and 4.5% glutaraldehyde. Following extensive washing,
the tissue was incubated in 2% osmium tetroxide and dehydrated
in ethanol. Embedded in Epon resin, ultrathin sections (74 nm)
were prepared, integrated onto grids and contrasted with uranyl
acetate and citrate. Images were taken with a transmission elec-
tron microscope (Zeiss, Oberkochen, Germany).
2.14. Immunoblot
The apical hydrogels were lysed in 480 ml RIPA plus 20 ml pro-
tease inhibitor (Roche, Mannheim, Germany). As a control,
200 mg human foreskin and a HFF-1 cell suspension (4 * 106 cells
per ml) were included in the analysis. Following, the ice-cooled
samples were ultrasonic treated for three cycles of each 10 s
including a break of 20 s (Branson SSE-1, VWR, Darmstadt, Ger-
many). Following, three freeze–thaw cycles between liquid nitro-
gen and 37 °C were performed. The supernatant was collected
after centrifugation at 10.000  g0 for 20 min. For a reducing
SDS-PAGE, 20 mg protein per collagen model, 50 mg protein per fib-
rin model and 20 mg protein per control (human foreskin and HFF-
1) were applied. Separation gels consisting of 5% acrylamide for
Collagen I (140 kDa) and 10% acrylamide for ß-actin (43 kDa) were
produced. To detect anti-human collagen I antibody (AF6220, R&D
systems, Wiesbaden, Germany) and anti-human ß-actin antibody
(3700, Cell Signaling Technologies, Leiden, Germany) were incu-
bated in a 1:1000 dilution over night at 4 °C on produced blots. A
HRP-coupled secondary antibody (Dianova, Hamburg, Germany)
was incubated for 1 h at room temperature in a 1:10000 dilution
on the membrane. Following, the antibody-complexes were
detected by chemiluminescence detection kit (Thermofisher,
Dreieich, Germany). The images were taken on a FluorochromQ
detection system (ProteinSimple, San Jose, USA). The images were
converted to 8-bit grey scale and the background was equally sub-
tracted in ImageJ. To calculate the area under the histogram, the
western blot analysis tool was applied. The relative density of col-
lagen I and ß-actin were calculated as a quotient to the reference
value of HFF-1.
6 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx

2.15. Statistical analysis of continuous donor-independent data
By Shapiro-Wilk test, the continuous donor-independent data
was identified as not-normally distributed. For non-normal dis-
tributed data, a Kruskal-Wallis-ANOVA followed by a post-hoc
Man-Whitney-Test was applied to compare the data groups. For
all quantitative data, a significance level was defined by a p-
value of  0.05. All statistical tests were performed with the soft-
ware OriginPro 9.1G (OriginLab Corporation, Northampton, US).
On glass the surface-adherent macrophages expressed the M2-
phenotypic marker CD163 [25,26] as well as the intercellular adhe-
sion molecule CD54 (Fig. 2A). After removing the fibrin hydrogel
and to assess the ratio of surface-adherent cells, the energy unit
ATP was quantified on all tested surfaces (Fig. 2B). Compared to
the culture on glass and PTFE, macrophages’ viability was signifi-
cantly increased on titanium (p-value  0.05, statistical tests were
described in the method section; collagen model: 23 ± 4 * 104 rel-
ative light units; fibrin model: 25 ± 3 * 104 relative light units).

2.16. Principal component analysis
The multi-parametric quantitative data were reduced by a prin-
cipal component analysis and the data matrix was thereby proven
on classification of the readout factors and the experimental
groups. To demonstrate the discriminatory power between PTFE
and titanium, the cocultures were solely included in the analysis.
Furthermore, the collagen models and the fibrin models were ana-
lyzed separately. For the analysis, the data matrix was centered to
the mean and scaled to the standard deviation. This allowed inde-
pendently from the measurement range and the measured units a
similar weighting of all measured parameters. Proceeding, the gen-
erated data matrix was reduced by the principal component anal-
ysis applying the software UnscramblerX (Camo, Oslo, Norway).
3. Results
Here, we present a model system to study in vitro the effect of
biomaterials on inflammation and fibrosis. This test model is a
modular approach consisting of macrophages and fibroblasts in a
3D collagen or fibrin matrix (see Fig. 1).
3.1. After a culture of 13 days, vital macrophages accumulated on
materials’ surface and integrated in the stacked fibrin clot (Fig. 2)
Surface-adherent macrophages sense the foreign body, respond
to it and translate the signal in the secretion of soluble mediators.
3.2. Surface exposure showed material-dependent coagulation of
plasma and fibrin clot formation (see Fig. 3A)
After tissue injury, the surface contact of blood plasma activates
the coagulation cascade. Thereby a fibrin clot is deposited on the
material [27]. The exposure of materials’ surface to blood plasma
was replicated in our in vitro model, by the induction of coagula-
tion, adding calcium to citrated plasma (see Supplement S3) [28].
By supporting coagulation, the inclusion of thrombin improved
standardization and the long-term stability of the fibrin clot (see
Supplement S4). Fibrin clot deposition, assessed by wet weight,
discriminated between biomaterials. The wet weight of the fibrin
clot increased significantly after contact to titanium in comparison
to glass and after contact to PTFE in comparison to titanium
(Fig. 3B). PTFE surfaces induced a significant (p-value  0.05)
higher fibrin clot formation (392 ± 55 mg for the collagen model
or 336 ± 49 mg for the fibrin model) than the exposure to glass
and titanium. In contrast, exhibiting a weight of 134 ± 16 mg for
the collagen model and 127 ± 16 mg for the fibrin model, the
plasma-derived fibrin clot formed in response to glass was signifi-
cantly lower (p-value  0.05). As demonstrated by the collagen
models versus the fibrin models, the apical compartment had no
influence on the fibrin clot formed on the surface. Interestingly,
the fibrin clot deposited after the monoculture of macrophages
(collagen model: 376 ± 48 mg; fibrin model: 368 ± 51 mg) was
circa 3-fold higher compared to respective cocultures, demonstrat-
ing the cells’ effect. When analyzing the material-exposed fibrin

Fig. 2. On the surface, macrophages viability was confirmed by histology and ATP-quantification. (A) On glass surfaces, the intercellular adhesion molecule (CD54, green) as
well as the M2-phenotypic marker CD163 (red) confirmed the presence of vital macrophages after 13 days of culture. The scale bar displays 50 mm and is valid for both
images. Cell nuclei were stained by DAPI. Fused cells with two nuclei were marked by a white triangle. (B) Viability of macrophages on the reference materials was quantified
by a luminescence-based ATP assay. Bar graphs represent mean values and standard errors of respective means. Explained by the analytical procedure, of five primary
macrophage donors (n = 5) two models (n = 2) were characterized in the analysis. The significance level was defined by a p-value of 0.05. A double asterisk (**) indicates an
experimental group, which differs significantly to all other experimental groups. Lines visualize significant differences between two experimental groups. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx 7

Fig. 3. Surface-exposed fibrin synthesis embeds vital macrophages. (A) A top view on the wound model system visualizes the basal part and the apical part integrated in the
biomaterial insert. The cell culture insert was removed to further characterize the surface-adjacent basal fibrin hydrogel. (B) Of five primary macrophage donors (n = 5), the
weight of the fibrin hydrogel on materials’ surface (basal weight) was assessed for two models (n = 2). A p-value of  0.05 defined significant differences. Hereby, a double
asterisk (**) illustrates an experimental group, which differed to all groups significantly. Printed lines mark significant differences between a pair of experimental groups. (C)
The scanning electron microscopic image (for further images in different magnification see supplemented Figs. S3 and S4) and (D) the Azan-stained light microscopic image
demonstrated the persistence of macrophages (marked with red arrrows and Azan provides dark red nuclei) in the surfac-exposed fibrin hydrogel (for further images see
supplemented Fig. S5). The scale bars depict in (C) 5 mm and in (D) 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

Fig. 4. Collagen and fibrin hydrogels in (A) the cell culture insert reconstruct the soft tissue surrounding an implant. Scanning electron microscopy visualized (B) a condensed
microstructure of the collagen hydrogels and (C) a loose matrix net of the plasma-derived fibrin hydrogels (for further images in different magnification see supplement
Figs. S3 and S4). Integrated fibroblasts formed filapodia, which were firmly connected with the loose matrix. The red arrows mark the interface between a fibroblast and the
extracellular matrix. The scale bar of 5 mm is valid for both images. (B) On day 13, the wet weight of the hydrogel (apical weight) in the cell culture insert was assessed for two
models (n = 2) of five primary donors (n = 5). A p-value of 0.05 characterizes a significant difference. A double asterisk (**) shows an experimental group, which differs to all
other groups significantly. Lines mark significant differences between two groups. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

clot, macrophages were directly located on the loose fiber net 3.4. After the coculture of macrophages and fibroblasts in fibrin
(Fig. 3C). A cross section highlighted macrophages’ accumulation models, a higher secretion of IL-1ß, IL-6 and IL-8 (Fig. 5A–C) was
at the interface of the clot (Fig. 3D). assessed in the supernatant than after the coculture in collagen models

3.3. The collagen hydrogel (Fig. 4A) formed dense connective
structures, whereas the plasma-derived fibrin clot consisted of a loose
matrix net (Fig. 4B and C)
Corresponding to our previous study (Jannasch et al., 2017), the
formation of dense collagen structures accompanied with a hydro-
gel contraction and concluded in a wet weight loss (Fig. 4D) [18]. In
this study, the wet weight was measured, to assess the weight
reduction in response to the biomaterials. Here, the collagen
hydrogels’ weight decreased after the culture on titanium
(326 ± 54 mg) with the lowest weight loss compared to PTFE
(248 ± 57 mg) with a moderate weight loss and glass
(111 ± 40 mg) with the highest weight loss. However, differences
between the reference materials PTFE and titanium were non-
significant (p-value = 0.2). Summarizing, the collagen model cov-
ered partially the expected higher weight loss in response to PTFE
compared to titanium. However, those effects were not significant.
A comparison of the cocultures in the two different matrix envi-
ronments revealed a 2-fold higher IL-1ß mean secretion after the
culture in the fibrin models than in the collagen models. Corre-
spondingly, the IL-6 mean level increased 3-fold after the culture
in the fibrin models compared to respective collagen models,
strengthening a consistent effect of the matrix environment. After
the coculture in fibrin models, a relative low increase of IL-8 (1.5-
fold) was detected compared to a culture in the collagen models.
Interestingly, the monocultures demonstrated the impact of fibrob-
lasts on the inflammatory milieu: fibroblasts integrated in a collagen
matrix secreted IL-6 (789 ± 171 pg per ml) and IL-8 (1574 ± 567 pg
per ml) moderately. Compared to that, fibroblasts embedded in a fib-
rin matrix secreted 200-fold higher IL-6 concentrations (16 ± 2 * 104
pg per ml) and 100-fold higher IL-8 concentrations (18 ± 3 * 104 pg
per ml). This influence of the extracellular matrix on the cell-
mediated inflammation strengthens a biomimetic reconstruction
of the wound microenvironment in a test system.

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8 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx

3.5. The cytokines IL-8 (Fig. 5B) and IL-1ß (Fig. 5C) increased from macrophages in the fibrin models amplified material-induced
PTFE with the lowest secretion, over glass with a moderate secretion to effects. However, compared to the collagen models, the observed
titanium with the highest secretion secretion patterns were not substantially modified.

In the supernatant of the fibrin models, the IL-1ß level differed
significantly (p-value  0.05): here the IL-1ß level doubled from
PTFE with 13 ± 5 pg per ml over glass (26 ± 5 pg per ml; p-
value = 0.02) to titanium (47 ± 9 pg per ml; p-value = 0.03). The
IL-6 secretion to the supernatant of the collagen models corre-
sponded to this pattern similarly. In case of IL-6, the levels
increased from 3 ± 0.7 * 104 pg per ml after culture on PTFE surfaces
to glass (5 ± 0.6 * 104 pg pro ml) and to titanium (6 ± 0.8 * 104 pg
pro ml). Interestingly, the combined culture of fibroblasts and
3.6. Titanium induced a significant secretion of the anti-inflammatory
cytokine IL-10 (Fig. 5D)
After the collagen exposed culture, the IL-10 response to tita-
nium (19 ± 2 pg per ml) was 3-fold higher than to PTFE (6 ± 2 pg
per ml). Consistently, the IL-10 level in the supernatant of the fib-
rin model was 2-fold increased after the culture on titanium
(11 ± 6 pg per ml) than on PTFE (5 ± 7 pg per ml). To conclude, as
previously shown, titanium induced the secretion of pro-

Fig. 5. The cells’ exposition to surfaces induced a material-dependent cytokine secretion. On day 13, for all three models (n = 3) the cytokines (A) IL-6, (B) IL-8, (C) IL-1ß, (D)
IL-10 and (E) TGF-ß1 were analyzed in the cell culture supernatant of each primary macrophage donor (n = 5). In the bar graph, mean values and standard errors of mean are
shown. The significance level is defined by a p-value of  0.05. Labeling with a double asterisk (**) describes an experimental group, which differs to all other groups
significantly. Labeling with a single asterisk (*) shows no significant differences between the monocultures, whereas significant differences to all other groups were found.
Lines indicate significant differences between two groups.

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 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx 9

inflammatory cytokines (IL-1ß, IL-6 and IL-8), as well as stimulated
an anti-inflammatory response (IL-10). This demonstrates, that the
cytokine ‘‘storm” to a biomaterial is complex, consisting of pro-
and anti-inflammatory mediators.
distinguished significantly (p-value = 0.02). To conclude, the
TGF-ß1 secretion covered expected higher values for PTFE com-
pared to titanium (collagen model 1344 ± 65 pg per ml; fibrin
model 1357 ± 237 pg per ml), whereas no significances were found
(p-value > 0.05).

3.7. TGF-ß1 in supernatant of the models exceeded the threshold in

controls without cells (Fig. 5E)
Relative high base levels were measured in the medium of the
controls without cells (collagen model: 934 ± 54 pg per ml; fibrin
model: 884 ± 49 pg per ml). Compared to this threshold, all
cocultures showed a significant higher TGF-ß1 secretion
(p-value  0.05). Highest changes relative to the base level were
induced in response to PTFE: a 2-fold increase was detected after
both the culture exposed to the collagen models (1833 ± 241 pg
per ml; p-value of < 0.001) and the culture exposed to the fibrin
models (2002 ± 348 pg per ml; p-value < 0.001). However, high
variations after the culture on PTFE explained non-significant
differences to the other materials (p-value > 0.05). Solely, after
the culture in the collagen models, TGF-ß1 levels between glass
(1664 ± 122 pg per ml) and titanium (1344 ± 65 pg per ml)
3.8. The complex profile of soluble mediators translated in (I) an
accumulation of extracellular matrix as well as (II) the fibroblasts’
proliferation
Addressing two central mechanisms during fibrosis, the extra-
cellular matrix accumulation and the fibroblasts’ proliferation, a
higher (I) collagen I synthesis (Fig. 6A) as well as a higher (II)
ß-actin (Fig. 6B) proportion was observed after the culture on PTFE
compared to titanium. In the collagen model, a relative collagen I
density of 59 ± 19% (Fig. 6C) was revealed after the culture on tita-
nium. Whereas, a collagen I density of 76 ± 19% was shown after
the culture on PTFE surfaces. In the fibrin model, the relative colla-
gen I density (8 ± 3%) was lower after the culture on titanium com-
pared to the culture on PTFE (14 ± 5%). Ultrastructural images
confirmed the newly synthesized collagen in a collagen model

Fig. 6. Relative expression of collagen I and ß-actin in the wound model. (A) The synthesis of collagen I (140 kDa) was quantified by western blots, detecting the pro-collagen,
the intermediary cleavage products and the final mature peptide of the collagen-a1(I)-chain. (B) ß-actin (43 kDa) was analyzed as a parameter for cell density. Per primary
macrophage donor, one model was characterized (n = 5) – blots of three replicates are presented in the figure. For (C) collagen I and (D) ß-actin, the area under the respective
gray scale histogram allowed an evaluation of the relative expression density, calculated as a ratio compared to HFF-1 (results are shown as a bar graph). In the analysis, 20 mg
protein per collagen model and 50 mg protein per fibrin model were applied. By transmission electron microscopy, ultrastructural analysis revealed newly synthesized
collagen in (E) a collagen model and (F) a fibrin model. (I) wound models and (II) single matrix models without cells were compared after culture on glass. Each scale bar
depicts 1 mm. The red arrows mark newly synthesized collagen bundles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

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10 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx

(Fig. 6E) compared to a fibrin model (Fig. 6F). Consistently, the con-
stitutively expressed structure protein ß-actin (Fig. 6D) revealed a
higher relative cell proliferation after the PTFE-exposed culture in
the fibrin model (37 ± 12%) compared to titanium (17 ± 9%). In
the collagen models, the relative cell density increased from
3 ± 2% in response to titanium surfaces to 12 ± 9% after the culture
on PTFE.
fibroblast monocultures consisted of loose collagen structures
(Fig. 8A). Beyond, material-exposed macrophages stimulated the
fibroblasts in the collagen to synthesize a dense fibrous tissue.
Whereas, after the culture in fibrin, the PTFE surfaces increased
the cell proportion (Fig. 8B). Summarizing, the histological analysis
strengthened the conducted relative quantification of collagen I
and cells’ density (Fig. 6).

3.9. Histological analysis confirmed an accumulation of extracellular 3.10. A principal component analysis showed the high discriminatory
matrix and fibroblasts’ proliferation power of both models (Fig. 9)

To illustrate the tissue structure and its composition, histologi-
cal sections were stained (Figs. 7 and 8). By a highly cross-linked
structure, collagen III stabilizes tissue defects and constitutes up
to 40% of the total pro-collagen during wound healing [29,30].
The collagen model exhibited a relative low collagen III synthesis
and showed no material dependency (Fig. 7A). In contrast, a strong
collagen III synthesis was shown after the PTFE-exposed culture in
the fibrin model (Fig. 7B). During wound healing, the thin and
highly elastic collagen III matrix is replaced by thicker organized
collagen I bundles and thereby the tissue is regaining tensile force
[31,32]. After 13 days in culture, the cell-free models as well as the
Previously developed multi-parametric comparative models
assumed significant differences between the experimental groups
as a discriminatory threshold (see Jannasch et al., 2017) [17]. In
this study, both wound models were partially characterized by
emerging trends. This finally hampered the interpretation of the
complex data and afforded alternative modelling techniques than
comparative statistical modelling. The principal component analy-
sis allows in general a reduction of multi-parametric data matrices,
and thereby facilitates a grouping of data endpoints and readout
parameters (for details on the analytical procedure see the method
section). A linear combination of all primary readout variables

Fig. 7. In the wound model, fibroblasts synthesized collagen III as an intermediary wound matrix. A histological collagen III staining was performed on one set of models. (A)
On day 13, a slight synthesis of collagen III was observed in the collagen models. (B) Whereas, in response to PTFE surfaces the fibrin models showed a high collagen III
synthesis. The scale bars depict 100 mm and are valid for all images.

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 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx 11

Fig. 8. Azan-stained sections visualize structure and composition of the tissue. (A) An increased collagen density (stained in blue) was detected in the collagen models. (B)
Whereas, fibroblasts in a plasma-derived fibrin matrix (stained in red) showed different proliferation ratios in response to the biomaterials. The scale bars depict 100 mm and
are valid for all images. The cyan mean intensity (CMI) was quantified by image analysis assessing the collagen density. For the respective histological sections, calculated
CMI ± SD values are shown in the right corner. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

determines the first principal component axis, which discriminates
the data matrix in direction of the highest variance. According to
that, the second principal component axis is orthogonally orien-
tated to the first principal component axis and discriminates the
data matrix subsequently by declining variance. For the collagen
models, the first two principal components explained 56% of the
data variance (Fig. 9A). Here, the first principal component exhib-
ited with variance of 30% a noisy signal, whereas the second prin-
cipal component discriminated with a variance of 26% between the
references PTFE and titanium. After the culture on PTFE surfaces,
the models projected in the positive axis intercept of the second
principal component. Whereas after the culture on titanium, the
models mapped in the negative axis intercept. In analogy, the prin-
cipal component analysis of the fibrin model exhibited a variance
of 51% for the first two principal components (Fig. 9A). With a vari-
ance of 31%, the first principal component depicted material char-
acteristics, allowing a grouping of the reference materials PTFE and
titanium. Negative values on the first principal component axis
were obtained for the model projections after the culture on PTFE
surfaces. In comparison, after the culture on titanium surfaces, the
models were assigned to positive values on the first principal com-
ponent axis. Additionally, the loading of all single readout param-
eters on the principal component plane visualized their
contribution to the discriminatory capacity of the models
(Fig. 9B). Factors characterizing a high fibrotic potential of a bioma-
terial, such as the surface-exposed fibrin synthesis (apical), the de
novo collagen I synthesis (collagen) and fibroblasts’ proliferation
(ß-actin) as well as the secretion of the pro-fibrotic growth factor
TGF-ß1, are illustrated as a purple area in Fig. 9B. All those fibrotic
readout factors projected after the culture on PTFE surfaces in a
similar axis intercept as the model projections. Retrospective, a
higher plasma-derived fibrin synthesis was observed on PTFE sur-
faces in both models (see comparison Table 2). Additionally, the
collagen I synthesis and fibroblasts’ proliferation and the TGF-ß1
secretion were increased after culture on PTFE surfaces (Table 2).
The consistent projection of all those fibrotic readout factors and
the models on the respective axis intercept confirmed the fibrotic
characteristics of PTFE. In contrast, parameters such as the
surface-exposed accumulation of macrophages (ATP), the secretion
of inflammatory cytokines (IL-1ß, IL-6, IL-8 and IL-10) and a stable
weight of the apical hydrogel are visualized as a green area on the
principal component plane (Fig. 9B). The loading of those readout

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12 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx

Fig. 9. Principal component analyses of the wound models. (A) The projections for (I) the collagen models and for (II) the fibrin models, in the principal component plane are
shown. (B) The loadings of the single readout parameters in the principal component plane visualize the contribution of those factors to the discriminatory capacity of (I) the
collagen models and (II) the fibrin models. To proof the discriminatory power of the wound models, the analysis comprised one model per primary blood donor (n = 5).

Table 2
The principal component analysis included all quantitative readout parameters, which characterized the wound models. The presented table summarizes the relative proportion
of the quantitative readout parameters for PTFE in comparison to titanium. Significant differences between PTFE and titanium (p-value  0.05) are presented with a star.

(I) collagen model (II) fibrin model

* *
viability of surface-adherent macrophages (ATP) PTFE < titanium PTFE < titanium
* *
material-exposed fibrin synthesis (basal weight) PTFE > titanium PTFE > titanium
connective tissue reduction (apical weight) PTFE < titanium PTFE < titanium
*
IL-6 PTFE < titanium PTFE = titanium
* *
IL-8 PTFE < titanium PTFE < titanium
* *
IL-1ß PTFE < titanium PTFE < titanium
* *
IL-10 PTFE < titanium PTFE < titanium
TGF-ß1 PTFE > titanium PTFE > titanium
collagen I synthesis (collagen) PTFE > titanium PTFE > titanium
fibroblasts proliferation (ß-actin) PTFE > titanium PTFE > titanium

parameters on the respective principal component intercept corre-
sponded to the model projections after the culture on titanium and
confirmed their influence on titanium’s evaluation. Most striking,
the established wound model was the first in vitro system linking
titanium’s characteristics to a high cytokine response and a high
material-associated cell viability (see Table 2). Summarizing, the
parameters’ loading matched for both models with respective
model projections on the principal component plane. Most impor-
tant, the higher fibrotic reaction in response to PTFE correlated to
in vivo studies [21,22] and to our previous published comparative
statistical model [17].
4. Discussion
Already a thin fibrous capsule hinders the contact of an implant
to functional tissue and thereby influences the therapies’ success.
This emphasizes the importance to assess biomaterial characteris-
tics sufficiently prior its use as an implant [33]. The study pre-
sented here addresses the clear need of in vitro tests, which
mimic a transient exposure of biomaterials to blood components
and resemble the soft tissue reaction in a long-term culture [8,34].
Bleeding occurs under all surgical procedures and exposes the
implant directly to blood fluid. This was the rational to establish
a model that mimics the contact to blood plasma. In our approach,
applied pooled plasma mitigated individual factors (e.g. coagula-
tion efficiency) and thereby reduced needed replicates. As a source
of coagulation factors, complement factors, platelets and fibrino-
gen, comprised plasma might allow further studies on
biomaterial-induced coagulation or complement activation.
Beyond the contact to blood plasma integrates our model mono-
cytes/macrophages as representative key players during the for-
eign body reaction. The study of Higgins et al. (2009) supported
our decision by showing macrophages and foreign body giant cells

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 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx 13

Table 3
Summary of pre-clinical and clinical studies on the response to PTFE and titanium after implantation. Based on this retrospective evaluation, the in this study developed in vitro
model system was validated.

Author (Year) Material Design Endpoint

[Ref]
Ungersböck [20] titanium versus pre-clinical study [canine, tibia Compared to stainless steel, the fibrous capsule developed around titanium was thinner
stainless steel implant] and showed lower inflammatory cell numbers.
Shannon [73] titanium versus pre-clinical study [rat, Between titanium and stainless steel no differences were observed in capsule thickness
stainless steel subcutaneous implant] and cell response. The qualitative characterization of titanium revealed a lower density
and a loose deposition of fibrous tissue.
Suska [19] titanium versus pre-clinical study [rat, In comparison to copper, titanium was surrounded by a thinner fibrous capsule and
copper subcutaneous implant] lower inflammatory infiltrates.
Banovetz [74] titanium versus clinical two-year-follow up study For titanium implants a failure rate of nine of 69 (13%) and for stainless steel implants a
stainless steel failure rate of two of 200 (1%) was observed.
Nautiyal [75] titanium clincal study [human, miniplates Titanium particles in peri-implant tissue, fibrosis, necrosis and inflammation correlated
used for maxillofacial fractures] to the patient’s pain, finally influencing implant failure.
Slosar [76] titanium clinical study [human, bone Titanium shows after implantation a good fusion with bone tissue.
implant]
Thomsen [21] titanium versus pre-clinical study [rat, abdominal The titanium implant was directly connected to the adjacent soft tissue. In contrast the
PTFE wall implant] PTFE implant was encapsulated by fibrous tissue.
von Recum [22] PTFE versus pre-clinical study [dog, aorta patch] Polyurethan was encapsulated by a fluide cyste, whereas on PTFE surfaces a moderate
polyurethane adherent fibrosis developed.
Trumpy [77] PTFE, hard and pre-clinical study [human, All materials induced the formation of a fibrous capsule, which expression declines in
soft silicone subcutaneous implant] order from soft silicone to PTFE and hard silicone.
Williams [78] PTFE clinical study [human, vascular The cumulative patency at 2 years was 29% falling to 19% at 4 years.
grafts for lower limb ischaemia]
Truswell [79] PTFE clinical study [human, facial soft The low-porosity expanded PTFE demonstrated a significant shrinkage and migration.
tissue augmentation]
Ferreira [80] PTFE clinical study [human, Teflon materials appeared to fail structurally and to cause a foreign body giant cell
temporomandibular joint alloplastic reaction.
implants]

localized directly on the surface, whereas other immune cells such
as B- and T-lymphocytes exhibited a broader range and distance
from the implant surface [35]. Even though the limitation of
in vitro system to specific cells and their function, the strong reli-
ance on the gold standard, and a direct translation of immunolog-
ical data from animals to humans should be critically scrutinized
[8,36,37]. For instance, macrophages from various species differ
in their phagocytosis, chemotaxis and cytotoxic sensitivity [38].
This low comparability emphasizes the high impact to integrate
monocytes from human blood in our in vitro models [11]. To main-
tain long-lived macrophages, monocytes were differentiated by
exogenous stimulation with M-CSF [25,39,40], M-CSF and GM-
CSF in the local plasma milieu [41–43] and the synthesis by inte-
grated fibroblasts [44]. The applied cell culture insert divided our
system and thereby supported, as in vivo observed, the persistence
and accumulation of macrophages on the surface. Others have
shown that a direct combined culture with fibroblasts hampers
the identification of vital macrophages [14–16], which in turn
excels our divided approach.
In comparison to animal studies, which entail cost and time
effort and often generate only a slight mechanistic insight, an
in vitro model provides a defined environment to test cellular
and molecular processes specifically [45]. In particular, the contin-
uous interface between the biomaterial and the key players during
a foreign body reaction established in the 3D models is of high rel-
evance. This continuous approach might be applied to track under
real time conditions cell phenotypes (adherence, phagocytosis,
proliferation, contraction, marker expression) and molecular pro-
files (e.g. cytokines, a-SMA). Finally, this will help to generate a
mechanistic understanding of the fibrotic response to biomaterials.
Furthermore, the equipment of implants with sensors (e.g. elec-
trodes) [46] or actuators (e.g. drug release) [47] increases the
requirement of a continuous characterization such as studies on
durability, stability and degradation. Most outstanding, our models
might be applied for a continuous evaluation of complex active
implants.
However, the acceptance of in vitro tests often fails by a low cor-
relation to in vivo outcomes [48]. The relevance to evaluate biomate-
rials’ effects by our in vitro approach was proven by tests on PTFE and
titanium. A comparison of preclinical studies, summarizing the
fibrotic potential after implantation, and a derived ranking was
already described as a quality criterion for our previous in vitro
model [17]. To allow a more detailed analysis, we here additionally
enclosed clinical studies on PTFE and titanium (see summary of pre-
clinical and clinical studies Table 3). Summarizing, independent of
implant morphology and implantation site, both pre-clinical and
clinical in vivo studies identified titanium as low fibrotic, whereas
PTFE induces a moderate fibrosis. Most outstanding, the reaction
pattern obtained in vitro correlates with those previously reported
in vivo studies, underlining the potential of our test procedures. A
comparison of both model systems revealed a higher collagen I
deposition in the collagen models, whereas a higher pro-
inflammatory milieu consisting of IL-1ß, IL-6 and IL-8 was assessed
in the fibrin models. Nevertheless, relative tendencies between both
models were consistent, leading to a similar cluster formation in the
principal component analysis. Interestingly, our data supports the
relevance of both models either based on collagen or fibrin.
In general, all model systems – in vivo as well as in vitro –
address specific functions and exhibit specific limitations. Our cur-
rent in vitro model covers major key mediators of a fibrotic reac-
tion. Local effects, such as a degradation of a collagen gel
implanted in the highly vascularized heart muscle and a preserva-
tion of the former matrix structure under the skin [49], envision
further development towards a site-specific model. Beyond vascu-
larization is the regeneration after injury dependent on the home-
ostasis of the functional cells’ proliferation versus the connective
cells’ proliferation [9]. In vivo, the regenerative capacity of tissues
ranges from continuous cycles (skin, intestine) and response to
injury (liver, pancreas) to a low regeneration accompanied with a
fibrotic tendency (nerve, heart muscle) [50]. Current in vitro tissues
such as skin [51], intestine [52], liver [53], pancreas [54,55], ner-
vous system [56,57] and heart [58] are of high impact to combine
with our models. Such a tissue-specific approach might excel a
regenerative capacity, and thereby allow further insight on the bal-
ance between fibrosis and regeneration. However, the regeneration
is limited by the degree of injury, which goes along with a destruc-

Please cite this article as: M. Jannasch, S. Gaetzner, F. Groeber et al., An in vitro model mimics the contact of biomaterials to blood components and the
reaction of surrounding soft tissue, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2019.03.029
14 M. Jannasch et al. / Acta Biomaterialia xxx (xxxx) xxx
tion of extracellular structures [9]. Our current hydrogel-based
approach might be improved by applying a native extracellular
matrix (e.g. decellularized tissue such as small intestine) towards
a higher regenerative capacity [59].
Subsequent the development of a test procedure, it needs a val-
idation to be approved as an alternative practice [60,61]. Therefore,
the reliability and the relevance of the test for a specific purpose
must be demonstrated in a multi-phase study. Prior to this, addi-
tional quality criterions such as endotoxin analysis should be con-
sidered, to increase the robustness of the established protocols.
Then, a technology transfer within one laboratory and to other lab-
oratories should verify the reproducibility of the assay. Moreover,
the application domain should be extended by tests on new bioma-
terials and further references (alginate, silicone, stainless steel). In
alignment to a reasoned application of animals in science, test on
novel biomaterials can substantiate a direct comparison of
in vitro versus in vivo reactions that will further improve validation.
[62,63]
Even if it takes a long breath to gain a validated test procedure,
we showed here the feasibility to analyze specific biomaterial-
induced effects such as inflammation and tissue remodeling
in vitro. Further tests must now confirm, whether our approach
based on blood components is able to close or even diminish the
gap of missing human-based in vitro models. We hope that others
will now follow our test design to improve current development.
Author contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding sources
Our work was funded by the German Federal Ministry of Educa-
tion and Research; program NanoMatFutur; grant agreement num-
ber 13N12971 – ETface. All authors declare no competing financial
interests in relation to the work described.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.actbio.2019.03.029.
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