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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


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

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

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

2.1. Ethical clearance statement 2.4. Isolation of human monocytes and integration in surface-exposed
fibrin hydrogels
Granted by the ethics committee of the Julius-Maximilians-
University Wuerzburg (vote 182/10), five human blood samples Mononuclear cells were isolated from leukocyte concentrate by
were obtained under informed signed consent according to the ficoll gradient centrifugation (GE Healthcare, Freiburg, Germany).
ethical approval (University Hospital Wuerzburg, Germany). Fro- To purify monocytes from the generated cell fraction, a negative
zen human blood plasma was purchased from the Bavarian Red magnetic cell separation selecting T cells, NK cells, B cells, dendritic
Cross blood donation service (Blutspendedienst des Bayerischen cells, and basophils was performed (Miltenyi Biotec, Bergisch Glad-
Roten Kreuzes, München, Germany). As defined in the Declaration bach, Germany). Following the isolation, the obtained monocytes
of Helsinki, the experiments with blood products were conducted (1 * 106 cells per model) were directly integrated in a plasma-
in compliance with the rules for investigation on human subjects. derived fibrin gel. As previously described by others [23,24],
800 ml plasma was equally distributed on materials’ surface
2.2. Pooled plasma (7 cm2) and 100 ml cell suspension (1 * 107 monocytes per ml
coculture medium) were added (see Figs. S1 and S2 for the per-
Frozen citrate-buffered human plasma (n = 10) was thawed for formed medium tests with fibroblasts and monocytes). This cocul-
2.5 h at 37 °C. For each donor, equal volumes of the obtained ture medium consisted of RPMI (Gibco) supplemented with 10%
human plasma were pooled (n = 10) and stored at - 80 °C. For fur- fetal calf serum (Gibco), 1 mM sodium pyruvate (Gibco; supports
ther use the frozen plasma was thawed as previously described. energy metabolism via citric acid cycle), 100 mM ascorbic acid
phosphate (Sigma Aldrich, Muenchen, Germany; supports as a
2.3. Preparation of test materials cofactor collagen synthesis) and 160 mg per ml t-AMCA (Pfizer,
Muenster, Germany; see Fig. S4 for information on its intended
Titanium, glass, and PTFE samples were prepared as previously use as fibrin stabilizer). To construct cell-free fibrin hydrogels,
applied [17,18]. Briefly, by radio frequency magnetron sputtering 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 cium chloride solution. To construct cell-free hydrogels, 50 ml
reversed by adding 100 ml thrombin (20 enzymatic units per ml; coculture medium was applied.
Sigma Aldrich, Muenchen, Germany) in 150 mM calcium chloride
solution (Sigma Aldrich, Muenchen, Germany). To support fibrin 2.5.2. (II) collagen models
synthesis, this mixture was incubated for 30 min at 37 °C. 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-
2.5. Apical hydrogel with human dermal fibroblast cell line HFF-1 in a
burg, Germany). In addition, cell-free collagen hydrogels were gen-
cell culture insert
erated by mixing collagen solution in a 2:1 ratio with gel
neutralizer solution (90 mM HEPES, 0.1 mM chondroitin sulfate,
The fibroblast cell line HFF-1 (ATCC, Wesel, Germany) was
3% FCS in 2-fold-concentrated DMEM; pH8.5). Following a gelation
seeded in a density of 4 * 103 cells per cm2. The medium consisting
for 30 min at 37 °C, the cell culture inserts were positioned on the
of DMEM (Gibco, Carlsbad, USA) plus 10% fetal calf serum
material-exposed fibrin gel.
(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 2.6. Replicates and culture of the models
polycarbonate membrane bottom with pores of 0.4 mm in diame-
ter, (I) plasma-derived fibrin hydrogels and (II) collagen hydrogels Per primary monocyte donor (n = 5), three technical replicates
were constructed. All cell-based models comprised 2 * 105 HFF-1 (n = 3) were constructed on glass, titanium and PTFE. Additionally,
cells (passage 18) in a final volume of 500 ml. 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
2.5.1. (I) fibrin models second to third day. To boost differentiation in the first 6 days,
The plasma-derived fibrin hydrogel was compounded as macrophages were stimulated with M-CSF (40 ng per ml; Pepro-
described in previous section. In short, 400 ml plasma was mixed tech, New Jersey, United States). Based on previous experience,
with 50 ml cell suspension (4 * 106 cells per ml coculture medium) the models were cultured for 13 days to further analyze tissue
and 50 ml thrombin (20 enzymatic units per ml) in 150 mM cal- 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 5

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

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

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

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

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 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 A comparison of the cocultures in the two different matrix envi-
structures, whereas the plasma-derived fibrin clot consisted of a loose ronments revealed a 2-fold higher IL-1ß mean secretion after the
matrix net (Fig. 4B and C) culture in the fibrin models than in the collagen models. Corre-
spondingly, the IL-6 mean level increased 3-fold after the culture
Corresponding to our previous study (Jannasch et al., 2017), the in the fibrin models compared to respective collagen models,
formation of dense collagen structures accompanied with a hydro- strengthening a consistent effect of the matrix environment. After
gel contraction and concluded in a wet weight loss (Fig. 4D) [18]. In the coculture in fibrin models, a relative low increase of IL-8 (1.5-
this study, the wet weight was measured, to assess the weight fold) was detected compared to a culture in the collagen models.
reduction in response to the biomaterials. Here, the collagen Interestingly, the monocultures demonstrated the impact of fibrob-
hydrogels’ weight decreased after the culture on titanium lasts on the inflammatory milieu: fibroblasts integrated in a collagen
(326 ± 54 mg) with the lowest weight loss compared to PTFE matrix secreted IL-6 (789 ± 171 pg per ml) and IL-8 (1574 ± 567 pg
(248 ± 57 mg) with a moderate weight loss and glass per ml) moderately. Compared to that, fibroblasts embedded in a fib-
(111 ± 40 mg) with the highest weight loss. However, differences rin matrix secreted 200-fold higher IL-6 concentrations (16 ± 2 * 104
between the reference materials PTFE and titanium were non- pg per ml) and 100-fold higher IL-8 concentrations (18 ± 3 * 104 pg
significant (p-value = 0.2). Summarizing, the collagen model cov- per ml). This influence of the extracellular matrix on the cell-
ered partially the expected higher weight loss in response to PTFE mediated inflammation strengthens a biomimetic reconstruction
compared to titanium. However, those effects were not significant. 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 3.6. Titanium induced a significant secretion of the anti-inflammatory
significantly (p-value  0.05): here the IL-1ß level doubled from cytokine IL-10 (Fig. 5D)
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 After the collagen exposed culture, the IL-10 response to tita-
IL-6 secretion to the supernatant of the collagen models corre- nium (19 ± 2 pg per ml) was 3-fold higher than to PTFE (6 ± 2 pg
sponded to this pattern similarly. In case of IL-6, the levels per ml). Consistently, the IL-10 level in the supernatant of the fib-
increased from 3 ± 0.7 * 104 pg per ml after culture on PTFE surfaces rin model was 2-fold increased after the culture on titanium
to glass (5 ± 0.6 * 104 pg pro ml) and to titanium (6 ± 0.8 * 104 pg (11 ± 6 pg per ml) than on PTFE (5 ± 7 pg per ml). To conclude, as
pro ml). Interestingly, the combined culture of fibroblasts and 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 distinguished significantly (p-value = 0.02). To conclude, the
an anti-inflammatory response (IL-10). This demonstrates, that the TGF-ß1 secretion covered expected higher values for PTFE com-
cytokine ‘‘storm” to a biomaterial is complex, consisting of pro- pared to titanium (collagen model 1344 ± 65 pg per ml; fibrin
and anti-inflammatory mediators. 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) 3.8. The complex profile of soluble mediators translated in (I) an
accumulation of extracellular matrix as well as (II) the fibroblasts’
Relative high base levels were measured in the medium of the proliferation
controls without cells (collagen model: 934 ± 54 pg per ml; fibrin
model: 884 ± 49 pg per ml). Compared to this threshold, all Addressing two central mechanisms during fibrosis, the extra-
cocultures showed a significant higher TGF-ß1 secretion cellular matrix accumulation and the fibroblasts’ proliferation, a
(p-value  0.05). Highest changes relative to the base level were higher (I) collagen I synthesis (Fig. 6A) as well as a higher (II)
induced in response to PTFE: a 2-fold increase was detected after ß-actin (Fig. 6B) proportion was observed after the culture on PTFE
both the culture exposed to the collagen models (1833 ± 241 pg compared to titanium. In the collagen model, a relative collagen I
per ml; p-value of < 0.001) and the culture exposed to the fibrin density of 59 ± 19% (Fig. 6C) was revealed after the culture on tita-
models (2002 ± 348 pg per ml; p-value < 0.001). However, high nium. Whereas, a collagen I density of 76 ± 19% was shown after
variations after the culture on PTFE explained non-significant the culture on PTFE surfaces. In the fibrin model, the relative colla-
differences to the other materials (p-value > 0.05). Solely, after gen I density (8 ± 3%) was lower after the culture on titanium com-
the culture in the collagen models, TGF-ß1 levels between glass pared to the culture on PTFE (14 ± 5%). Ultrastructural images
(1664 ± 122 pg per ml) and titanium (1344 ± 65 pg per ml) 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- fibroblast monocultures consisted of loose collagen structures
stitutively expressed structure protein ß-actin (Fig. 6D) revealed a (Fig. 8A). Beyond, material-exposed macrophages stimulated the
higher relative cell proliferation after the PTFE-exposed culture in fibroblasts in the collagen to synthesize a dense fibrous tissue.
the fibrin model (37 ± 12%) compared to titanium (17 ± 9%). In Whereas, after the culture in fibrin, the PTFE surfaces increased
the collagen models, the relative cell density increased from the cell proportion (Fig. 8B). Summarizing, the histological analysis
3 ± 2% in response to titanium surfaces to 12 ± 9% after the culture strengthened the conducted relative quantification of collagen I
on PTFE. 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- Previously developed multi-parametric comparative models
cal sections were stained (Figs. 7 and 8). By a highly cross-linked assumed significant differences between the experimental groups
structure, collagen III stabilizes tissue defects and constitutes up as a discriminatory threshold (see Jannasch et al., 2017) [17]. In
to 40% of the total pro-collagen during wound healing [29,30]. this study, both wound models were partially characterized by
The collagen model exhibited a relative low collagen III synthesis emerging trends. This finally hampered the interpretation of the
and showed no material dependency (Fig. 7A). In contrast, a strong complex data and afforded alternative modelling techniques than
collagen III synthesis was shown after the PTFE-exposed culture in comparative statistical modelling. The principal component analy-
the fibrin model (Fig. 7B). During wound healing, the thin and sis allows in general a reduction of multi-parametric data matrices,
highly elastic collagen III matrix is replaced by thicker organized and thereby facilitates a grouping of data endpoints and readout
collagen I bundles and thereby the tissue is regaining tensile force parameters (for details on the analytical procedure see the method
[31,32]. After 13 days in culture, the cell-free models as well as the 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 ponent axis. Additionally, the loading of all single readout param-
the data matrix in direction of the highest variance. According to eters on the principal component plane visualized their
that, the second principal component axis is orthogonally orien- contribution to the discriminatory capacity of the models
tated to the first principal component axis and discriminates the (Fig. 9B). Factors characterizing a high fibrotic potential of a bioma-
data matrix subsequently by declining variance. For the collagen terial, such as the surface-exposed fibrin synthesis (apical), the de
models, the first two principal components explained 56% of the novo collagen I synthesis (collagen) and fibroblasts’ proliferation
data variance (Fig. 9A). Here, the first principal component exhib- (ß-actin) as well as the secretion of the pro-fibrotic growth factor
ited with variance of 30% a noisy signal, whereas the second prin- TGF-ß1, are illustrated as a purple area in Fig. 9B. All those fibrotic
cipal component discriminated with a variance of 26% between the readout factors projected after the culture on PTFE surfaces in a
references PTFE and titanium. After the culture on PTFE surfaces, similar axis intercept as the model projections. Retrospective, a
the models projected in the positive axis intercept of the second higher plasma-derived fibrin synthesis was observed on PTFE sur-
principal component. Whereas after the culture on titanium, the faces in both models (see comparison Table 2). Additionally, the
models mapped in the negative axis intercept. In analogy, the prin- collagen I synthesis and fibroblasts’ proliferation and the TGF-ß1
cipal component analysis of the fibrin model exhibited a variance secretion were increased after culture on PTFE surfaces (Table 2).
of 51% for the first two principal components (Fig. 9A). With a vari- The consistent projection of all those fibrotic readout factors and
ance of 31%, the first principal component depicted material char- the models on the respective axis intercept confirmed the fibrotic
acteristics, allowing a grouping of the reference materials PTFE and characteristics of PTFE. In contrast, parameters such as the
titanium. Negative values on the first principal component axis surface-exposed accumulation of macrophages (ATP), the secretion
were obtained for the model projections after the culture on PTFE of inflammatory cytokines (IL-1ß, IL-6, IL-8 and IL-10) and a stable
surfaces. In comparison, after the culture on titanium surfaces, the weight of the apical hydrogel are visualized as a green area on the
models were assigned to positive values on the first principal com- principal component plane (Fig. 9B). The loading of those readout

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
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- This emphasizes the importance to assess biomaterial characteris-
sponded to the model projections after the culture on titanium and tics sufficiently prior its use as an implant [33]. The study pre-
confirmed their influence on titanium’s evaluation. Most striking, sented here addresses the clear need of in vitro tests, which
the established wound model was the first in vitro system linking mimic a transient exposure of biomaterials to blood components
titanium’s characteristics to a high cytokine response and a high and resemble the soft tissue reaction in a long-term culture [8,34].
material-associated cell viability (see Table 2). Summarizing, the Bleeding occurs under all surgical procedures and exposes the
parameters’ loading matched for both models with respective implant directly to blood fluid. This was the rational to establish
model projections on the principal component plane. Most impor- a model that mimics the contact to blood plasma. In our approach,
tant, the higher fibrotic reaction in response to PTFE correlated to applied pooled plasma mitigated individual factors (e.g. coagula-
in vivo studies [21,22] and to our previous published comparative tion efficiency) and thereby reduced needed replicates. As a source
statistical model [17]. of coagulation factors, complement factors, platelets and fibrino-
gen, comprised plasma might allow further studies on
biomaterial-induced coagulation or complement activation.
4. Discussion Beyond the contact to blood plasma integrates our model mono-
cytes/macrophages as representative key players during the for-
Already a thin fibrous capsule hinders the contact of an implant eign body reaction. The study of Higgins et al. (2009) supported
to functional tissue and thereby influences the therapies’ success. 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 rials’ effects by our in vitro approach was proven by tests on PTFE and
as B- and T-lymphocytes exhibited a broader range and distance titanium. A comparison of preclinical studies, summarizing the
from the implant surface [35]. Even though the limitation of fibrotic potential after implantation, and a derived ranking was
in vitro system to specific cells and their function, the strong reli- already described as a quality criterion for our previous in vitro
ance on the gold standard, and a direct translation of immunolog- model [17]. To allow a more detailed analysis, we here additionally
ical data from animals to humans should be critically scrutinized enclosed clinical studies on PTFE and titanium (see summary of pre-
[8,36,37]. For instance, macrophages from various species differ clinical and clinical studies Table 3). Summarizing, independent of
in their phagocytosis, chemotaxis and cytotoxic sensitivity [38]. implant morphology and implantation site, both pre-clinical and
This low comparability emphasizes the high impact to integrate clinical in vivo studies identified titanium as low fibrotic, whereas
monocytes from human blood in our in vitro models [11]. To main- PTFE induces a moderate fibrosis. Most outstanding, the reaction
tain long-lived macrophages, monocytes were differentiated by pattern obtained in vitro correlates with those previously reported
exogenous stimulation with M-CSF [25,39,40], M-CSF and GM- in vivo studies, underlining the potential of our test procedures. A
CSF in the local plasma milieu [41–43] and the synthesis by inte- comparison of both model systems revealed a higher collagen I
grated fibroblasts [44]. The applied cell culture insert divided our deposition in the collagen models, whereas a higher pro-
system and thereby supported, as in vivo observed, the persistence inflammatory milieu consisting of IL-1ß, IL-6 and IL-8 was assessed
and accumulation of macrophages on the surface. Others have in the fibrin models. Nevertheless, relative tendencies between both
shown that a direct combined culture with fibroblasts hampers models were consistent, leading to a similar cluster formation in the
the identification of vital macrophages [14–16], which in turn principal component analysis. Interestingly, our data supports the
excels our divided approach. relevance of both models either based on collagen or fibrin.
In comparison to animal studies, which entail cost and time In general, all model systems – in vivo as well as in vitro –
effort and often generate only a slight mechanistic insight, an address specific functions and exhibit specific limitations. Our cur-
in vitro model provides a defined environment to test cellular rent in vitro model covers major key mediators of a fibrotic reac-
and molecular processes specifically [45]. In particular, the contin- tion. Local effects, such as a degradation of a collagen gel
uous interface between the biomaterial and the key players during implanted in the highly vascularized heart muscle and a preserva-
a foreign body reaction established in the 3D models is of high rel- tion of the former matrix structure under the skin [49], envision
evance. This continuous approach might be applied to track under further development towards a site-specific model. Beyond vascu-
real time conditions cell phenotypes (adherence, phagocytosis, larization is the regeneration after injury dependent on the home-
proliferation, contraction, marker expression) and molecular pro- ostasis of the functional cells’ proliferation versus the connective
files (e.g. cytokines, a-SMA). Finally, this will help to generate a cells’ proliferation [9]. In vivo, the regenerative capacity of tissues
mechanistic understanding of the fibrotic response to biomaterials. ranges from continuous cycles (skin, intestine) and response to
Furthermore, the equipment of implants with sensors (e.g. elec- injury (liver, pancreas) to a low regeneration accompanied with a
trodes) [46] or actuators (e.g. drug release) [47] increases the fibrotic tendency (nerve, heart muscle) [50]. Current in vitro tissues
requirement of a continuous characterization such as studies on such as skin [51], intestine [52], liver [53], pancreas [54,55], ner-
durability, stability and degradation. Most outstanding, our models vous system [56,57] and heart [58] are of high impact to combine
might be applied for a continuous evaluation of complex active with our models. Such a tissue-specific approach might excel a
implants. regenerative capacity, and thereby allow further insight on the bal-
However, the acceptance of in vitro tests often fails by a low cor- ance between fibrosis and regeneration. However, the regeneration
relation to in vivo outcomes [48]. The relevance to evaluate biomate- is limited by the degree of injury, which goes along with a destruc-

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

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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