Biodegradable poly (lactic acid-co-glycolic acid) scaffolds as carriers for genetically-modified fibroblasts
Biodegradable poly (lactic acid-co-glycolic acid) scaffolds as carriers for genetically- modified fibroblasts
Tatjana Perisic 0 1 2
Ziyang Zhang 0 2
Peter Foehr 0 2
Ursula Hopfner 0 1 2
Kathrin Klutz 0 1 2
Rainer H. Burgkart 0 2
Alexei Slobodianski 0 1 2
Moritz Goeldner 0 2
Hans-GuÈ nther Machens 0 1 2
Arndt F. Schilling 0 1 2 3
0 Current address: Institute for Technology and Innovation Management, Hamburg University of Technology , Hamburg , Germany
1 Experimental Plastic Surgery, Department for Plastic Surgery and Hand Surgery, Klinikum rechts der Isar, Klinikum rechts der Isar, Technische UniversitaÈ t MuÈ nchen , Munich, Germany , 2 Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, Hubei , China , 3 Department of Orthopaedics and Sportsorthopaedics, Klinikum rechts der Isar, Technische Universit aÈt MuÈ nchen , Munich, Germany , 4 Johnson & Johnson MEDICAL GmbH , Norderstedt , Germany
2 Editor: HeÂlder A. Santos , Helsingin Yliopisto , FINLAND
3 Klinik fuÈ r Unfallchirurgie, Orthop aÈdie und Plastische Chirurgie, University Medical Center GoÈttingen , GoÈ ttingen , Germany
Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid) (PLGA) scaffolds (Vicryl & Ethisorb) as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days) of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF165) and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was partly supported by a
grant to Dr. Ziyang Zhang from National Natural
Science Foundation of China (Grant No. No.
81401538). This study was also partly supported
by an unrestricted the grant of Johnson & Johnson
Medical GmbH, Norderstedt, Germany. Johnson &
Johnson Medical GmbH provided support in the
form of salaries for author MG, but did not have
any additional role in the study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. The specific role of
this author is articulated in the `author
New generation non-viral gene delivery systems, such as nanoparticles, novel cationic lipids
and polymers, chemically coupling the nucleic acid to peptides and polymers [1±9] have
shown to overcome some of the limitations of viral gene transfer and offer advantages in use of
recombinant proteins [
]. On the other side, the localized delivery of genetically modified
cells themselves poses another major barrier for efficient and area-specific therapeutic protein
expression in gene therapy applications. Challenges regarding the reduction of off-target
effects remain to be addressed [
]. Thus, biodegradable synthetic polymer scaffolds may offer
a safe and effective option for targeted cell delivery.
Biodegradable scaffolds are routinely used as reinforcement material for various surgical
procedures, including hernia repair [
], tendon reconstruction [
], cranio-maxillo-facial surgery
or neurosurgery [15±18]. Synthetic polymers such as polylactic acid (PLA), polyglycolic acid
(PGA) and their copolymer poly(lactic acid-co-glycolic acid) (PLGA) are also highly useful for
the temporary management of pathologically altered tissue architectures including ligaments,
skin, vascular tissues and skeletal muscle [
]. The implanted scaffolds not only provide
structural support but also guide new tissue ingrowth and, importantly, are completely absorbed by
the body without the need for subsequent surgical removal. As an alternative to fully synthetic
polymer-based scaffolds, decellularized biological matrix material of xenogenic or allogenic
origin can be used [
]. Biological scaffolds exhibit a tensile strength comparable to that of
PLGAbased scaffolds and show superior collagen deposition and organisation [
]. Still, xenografts
and allografts have serious constrains due to limited availability, residual α-Gal-mediated
immunogenicity , as well as the risk of transmission of animal- or human-derived infectious agents
. Biodegradability of the matrix material is a crucial issue in the development of tissue
engineering scaffold structures. By utilizing easy manufactured off-the-shelf synthetic absorbable
polymers, complications associated with antigenicity and disease transmission can be eliminated.
Another important factor in scaffold-aided tissue regeneration is that this process is critically
dependant on efficient vascularization [24±28]. Moreover, local delivery of recombinant
angiogenic growth factors from scaffold materials presents a promising strategy in promoting
formation of a functional blood vessel network within the regenerated tissue [
]. However, the
short half-life of the recombinant proteins hinders the unleashing of their full angiogenic
potential. Given the essential role in reparation of damaged tissue, fibroblasts are particularly suitable
as cellular vehicles for delivery of angiogenic growth factors in tissue engineering  as well as
for gene therapy applications . Thus, in this study, we applied an expression vector for
vascular endothelial growth factor (VEGF165) as a model with which we studied the utility of
PLGAbased scaffolds as carriers for genetically modified cells. We used an established transfection
procedure on human Hs27 fibroblasts and tested their proliferation and morphology on two
PLGAbased scaffolds Vicryl and Ethisorb. The rationale behind using these scaffolds is that they are
already established in clinical use and PLGA has been shown to provide excellent conditions for
attachment, growth and motility of human skin fibroblasts [
]. Furthermore, we investigated
mechanical properties of the scaffolds seeded with cells over the short time frame of 9 days.
Finally, Vicryl and Ethisorb were compared in terms of their ability to serve as adhesion stratums
for VEGF165-overexpressing Hs27 cells and production of angiogenic VEGF165 protein.
Materials and methods
Cells and Vicryl and Ethisorb scaffolds
Human fibroblast cell line Hs27 (ATCC; CRL-1634) was cultured under standard condition
(37ÊC, 5% CO2) in Dulbecco's Modified Eagle Medium (DMEM) containing phenol red, stable
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glutamine and 4 g glucose/l (PAA, Coelbe, Germany). The culture medium was supplemented
with 10% fetal calf serum (FCS: heat inactivated FCS-Gold, PAA, Coelbe, Germany) and
antibiotic/antimycotic solution (AB/AM; PAA, Coelbe, Germany). DMEM containing FCS and
AB/AM solution is referred herein as growth medium. For this study, two types of absorbable
surgical scaffolds were used: Vicryl (VM 802) and Ethisorb (Patch Typ 6; ZVP 203) scaffold
(Ethicon, Johnson & Johnson Medical, GmbH, Norderstedt, Germany). The Vicryl scaffold
consists of polyglactin 910, which is a co-polymer of 90% glycolide and 10% L-lactide. Ethisorb
is also a polyglactin 910-based implantable scaffold and it consists mainly of polyglactin 910
and a small proportion of poly-P-dioxanon. The resorption time for polyglactin 910 is 45±60
days and for poly-P-dioxanon 90±180 days [
Cell growth on Vicryl and Ethisorb scaffolds
For both types of scaffolds, a cell suspension of 5 x105 Hs27 cells in 4 ml growth medium was
pipetted in 4-well rectangle plates (Thermo Fisher Scientific, Waltham, MA, USA). Vicryl and
Ethisorb patches were immediately placed inside. The scaffolds were incubated overnight in a
cell incubator under standard conditions on a horizontal shaker (Duomax 1030,Heidolph,
Germany). For controls, the scaffold was put in 4 ml growth medium without Hs27 cells and
handled the same way as the scaffold seeded with human fibroblast cells. To remove Hs27 cells
which did not attach to the scaffold, 16±24 hours after initial seeding, the scaffold with
adherent cells was transferred in new 4-well rectangle plates filled with 4 ml fresh growth medium
per well and further incubated under standard conditions. Proliferation rate, morphology, and
distribution of attached Hs27 cells, as well as the mechanical properties of the scaffold
populated with cells were examined 1, 3, 6 and 9 days after the seeding of cells.
Measurement of Hs27 cell proliferation activity on Vicryl and Ethisorb scaffolds
Cell proliferation activity was measured by ready-to-use colorimetric WST assay (Roche
Molecular Biochemicals, Basel, Switzerland). For that, all specimens were transferred in a new
rectangle 4-well plate with 3 ml fresh growth medium supplemented with WST solution and
incubated for 2 hours at 37ÊC. The assay principle is based upon the reduction of the
tetrazolium salt WST to formazan by cellular dehydrogenases. The generation of the dark yellow
coloured formazan is measured at 450 nm and is directly correlated to cell number. The
measurement was performed according to the manufacturer's instructions by using a microplate
reader (Mithras LB 940, Berthold Technologies GmbH, Germany).
Visualisation of morphology of Hs27 cells grown on Vicryl and Ethisorb scaffolds
In order to investigate the cell morphology, scaffolds seeded with Hs27 cells were fixed either
in 3% glutaraldehyde or in 3,7% formaldehyde. Two different methods for cell visualisation
were applied: Giemsa staining and scanning electron microscopy (SEM). For Giemsa staining,
formaldehyde-fixed specimens were stained for 10 min in a 1:20 pre-diluted Giemsa-solution
(PAA, Coelbe, Germany) and rinsed 3 times in Ultra pure water. Photos were taken with a
CCD-camera under the microscope (Nikon; Eclipse TE2000-S). For SEM, glutaraldehyd-fixed
scaffolds were dehydrated with graded ethanol, dried and sputtered with gold (Baltec SCD005;
40 mA; 80 s). Control scaffolds were handled the same way as cell-seeded scaffolds. SEM was
carried out using a Jeol-SEM-5400 (Eching) in Hi-Vac mode by applying an acceleration
voltage of 5 kV and detecting secondary electrons for imaging.
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Measurement of mechanical properties of cell-seeded and control Vicryl and Ethisorb scaffolds
The mechanical properties of Vicryl and Ethisorb scaffolds were tested during tensile testing.
A universal test system (zwicki1120, Zwick/Roell, Ulm, Germany) was used, equipped with a
2.5 kN load cell (Type KAF-Z class 0.1, A.S.T., Dresden, Germany). The samples were clamped
along their longer side in axial direction. To avoid shear stresses and torsional moments a
passive x-y-Mz-compensation tool was inserted between the upper clamp and the load cell. A
free length of L0 = 20 mm was defined as an elongation of 0%. Subsequently, the sample was
stretched at a constant speed of 10 mm/min until total failure of the sample. Tensile testing
was stopped automatically by the test system after a force drop of 40% occurred, with respect
to maximum force. Maximum force was determined from the highest force value of the
forcedisplacement graph. All mechanical results were summarized as mean values (mean) and
standard error of the mean (SEM).
Transfection of Hs27 cells with GFP and VEGF165 plasmids and cultivation on Vicryl and Ethisorb scaffolds
For transfection experiments, human fibroblasts were cultivated under standard conditions in
growth medium in T175 flasks. Upon reaching confluence, 5 x 106 cells were transfected with
20 μg plasmid DNA coding for green fluorescent protein (GFP) provided in the fibroblast
transfection kit (Lonza, Cologne, Germany). The transfection was performed by using
Nucleofector transfection apparatus (Lonza, Cologne, Germany) and DT130 transfection program.
After 16 hours cultivation, the cells were trypsinized, washed and transferred into tubes, which
contained pre-cutted Vicryl or Ethisorb scaffolds (1,5 cm x 1,5 cm). The tubes were gently
rotated in an incubator for additional 16 hours. Subsequently, the scaffolds were taken out and
carefully laid in another plate with fresh growth medium and further cultivated for a defined
period (1, 3, 6 and 9 days) under standard conditions. By using the same protocol, human
fibroblasts were transfected with 20 μg VEGF165 plasmid DNA [
] and grown on Vicryl
or Ethisorb scaffolds. After 1, 3, 6 and 9 days, the supernatant was collected, temporarily stored
at -20ÊC and used for subsequent measurements of VEGF165 protein concentration.
Detection of intracellular GFP expression and extracellular VEGF165 protein levels
The temporal expression of GFP protein in Hs27 cells was visualised by using the fluorescent
microscope (Zeiss Axio Observer.A1, Software AxioVision). The level of VEGF165 protein
secreted from Hs27 cells grown on Vicryl and Ethisorb scaffolds was determined by using
Human VEGF Quantikine ELISA Kit (R&D Systems) according to the manufacturer's
instructions. The absorbance at 450 nm was measured with a microplate reader (Mithras LB 940,
Berthold Technologies GmbH, Germany).
The data for the proliferation activity and tensile testing are from single experiments with five
technical replicates. The statistical comparison was performed by two-way ANOVA with
Tuckey's post-hoc testing. ELISA experiment was performed three times with three technical
replicates. The statistical testing was done by two-way ANOVA followed by Sidak's multiple
comparison tests. For all statistical tests, differences among means were considered significant
when the p value was < 0.05.
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Proliferation of Hs27 cells on Vicryl and Ethisorb scaffolds
Hs27 cell proliferation could be observed on images of cell-seeded Vicryl and Ethisorb
scaffolds captured with the light microscope in the course of 9 days (Fig 1). At day one, the cells
were scarcely distributed on both types of scaffolds. After 9 days of cultivation, most of the
surface of both scaffolds was covered with Hs27 cells. Furthermore, on the images obtained by
light microscopy, it is visible that the textile structure of Vicryl scaffold is woven and contains
small pores whereas that of Ethisorb is nonwoven. This implicates the difference in the surface
area between Ethisorb and Vicryl scaffolds. In order to quantify the changes in cell number
observed by the light microscope, the WST assay was applied. As seen on Fig 2, the increase in
cell number is reflected by an increase in dehydrogenase activity over the time course of the
experiment. When compared to Vicryl scaffolds, the Hs27 cells on Ethisorb scaffolds showed
higher values at all time points, as measured by an increase in optical density of formazan dye
at 450 nm, probably caused by the increased surface area available to the cells. The parallel
increase of the absorbance in both groups over time reflects a similar rate of proliferation of
the attached cells on both scaffolds.
Morphology of Hs27 cells on Vicryl and Ethisorb scaffolds
A suitable way to investigate the topography and morphology of rough surfaces is SEM. In this
study, it was used for a close inspection of morphology of adherent cells and the physical state
of the scaffold fibers. The SEM did not show any obvious signs of degradation of the polymer
fibers of Vicryl and Ethisorb scaffolds (with or without attached Hs27 cells) after 9 days in
culture (Fig 3). Furthermore, there was a nearly confluent layer of cells visible on both scaffolds at
day 9. The cells grown on scaffolds have maintained their typical polygonal flattened shape.
The SEM also demonstrated that the attached cells are predominantly located between the
polymer fibers of the scaffolds, bridging the inter-fiber gaps with multiple cell protrusions.
Mechanical properties of Vicryl and Ethisorb scaffolds populated with and without Hs27 cells
Next, the mechanical strength of Vicryl and Ethisorb scaffolds (with and without attached
Hs27 cells) was investigated using tensile testing (Fig 4A and 4B).
The presence of cells did not affect the mechanical behaviour of the scaffolds for both Vicryl
and Ethisorb at any time points investigated (Fig 4C).
Furthermore, the mean values of the maximal tensile force for Vicryl scaffolds (with and
without cells) only slightly decreased over the course of the 9 days. On the other side, the
maximal tensile force of the Ethisorb scaffolds (with and without cells) dropped by almost 50% at
day 9. Under the used experimental conditions, Ethisorb scaffolds had significantly higher
tensile strength at days 1, 3 and 6 compared to Vicryl.
Transient GFP and VEGF165 expression in Hs27 cells grown on Vicryl and Ethisorb scaffolds
Finally, the difference in the level of GFP expressed in Hs27 cells and the VEGF165 protein
secreted from Hs27 cells grown on Vicryl and Ethisorb scaffolds was investigated. By using the
GFP expressing plasmid a general transfectability of the Hs27 cells was tested and whether
they adhere on the scaffolds upon transfection. Both GFP and VEGF165 transfected cells
successfully adhered on the surface of the scaffolds (Fig 5A and 5B). For both Vicryl and Ethisorb,
the expression of GFP protein was the highest on day 1 and day 3 and then gradually decreased
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Fig 1. Visualisation of Hs27 cell growth on Vicryl and Ethisorb scaffolds. During the co-cultivation period of 9 days, Hs27 fibroblast cells on Vicryl (A)
and Ethisorb (B) scaffolds were investigated at four different time points by means of light microscopy with 100-fold magnification. In the upper row of each
panel, the scaffolds containing no cells (control) are presented. The fibroblasts were visualized with Giemsa staining and can be seen in the images of the
bottom row of each panel as small blue dots.
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Fig 2. Comparison of proliferation activity of Hs27 cells grown on Vicryl and Ethisorb scaffolds. Hs27 cells were seeded on Vicryl and
Ethisorb scaffolds and cultivated for a period of 9 days. At day (d) 1, 3, 6 and 9 the proliferation activity was measured by means of colorimetric WST
assay. The results of one experiment are presented as means of absorbance at 450 nm. The error bars represent SEM of five technical replicates
(n = 5; two-way ANOVA comparing proliferation activity of cells between Vicryl and Ethisorb for each time point: ****p<0.00005).
on day 6 and day 9 Similarly, the peak of VEGF165 protein expression from cells grown on
Vicryl and Ethisorb scaffolds was reached on day 3 with gradual decrease in the course of next
6 days (Fig 6). At all investigated time points, the level of VEGF165 protein secretion from
Hs27 cells grown on Ethisorb scaffolds was significantly higher than the level of VEGF165
protein secretion from cells grown on Vicryl scaffolds.
Due to significant advances in recombinant DNA technology, cell-based gene therapy is
increasingly recognized as an ideal tool to deliver therapeutic proteins to target sites. A typical
way of administering genetically modified cells is the direct injection of cells in target areas.
However, this mode of administration is accompanied by the spread away from the injection
site. The spreading of transfected cells can reduce vector levels at the target site and also lead to
low-grade systemic spread in off-target regions [
]. There are different strategies in
addressing those concerns. For example, in adoptive T cell therapy, new generation of chimeric
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Fig 3. Analysis of scaffold fibers and Hs27 cell morphology by scanning electron microscopy. Hs27 fibroblasts
are grown on Vicryl and Ethisorb scaffolds and visualized at day 1, day 3 day6 and day 9 in culture by means of scanning
electron microscopy (Scale bars in the upon two rows in A and B are 100μm, in the lowest row are 25μm)
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Fig 4. Determination of mechanical properties for Vicryl and Ethisorb scaffolds with and without seeded cells.
The mechanical properties of Vicryl (A) and Ethisorb (B) scaffolds were measured by using a uniaxial test system. Meshes
were clamped along their long side with an initial length L0 of 20 mm. Maximum force values for the two types of scaffolds
(both with and without Hs27 cells) were measured in a single experiment at day (d) 1, 3, 6 and 9 and presented graphically
(C). All error bars attached to the mean values represent the SEM of five technical replicates (n = 5; two-way ANOVA
comparing maximum force values of Vicryl and Ethisorb scaffolds for each time point: ****p<0.00005).
antigen receptors called TRUCKs (T cells redirected for universal cytokine killing) are used as
vehicles to produce and release a transgenic product that accumulates in the targeted tissue
] and this strategy can potentially yield effective and localized therapeutic effects. Another
approach in targeted cancer gene therapy of solid tissues is based on local delivery of NK4
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Fig 5. Visualisation of GFP-expressing Hs27 cells adhered on Vicryl and Ethisorb scaffolds. 5 x 106
Hs27 cells were electroporated with 20 μg GFP plasmid by using Nucleofector transfection apparatus. After a
short period in culture, transfected cells were transferred on scaffolds. The expression of GPF protein in Hs27
fibroblast grown on Vicryl (A) and Ethisorb (B) scaffolds was recorded at day 1, 3, 6 and 9 by a fluorescent
microscope with 100-fold magnification. In the upper row of each panel, the scaffolds containing no cells are
(hepatocyte growth factor antagonist) secreted from an NK4 gene-transduced oral mucosal
epithelial cell [
]. A similar technique by using cellular vehicle for delivery of bone
morphogenetic protein-2 has been used to promote bone regeneration [
]. Thus, genetically
transfected cells can be used for delivery of therapeutic cells to the intended area in the body.
In general, growing living cells on scaffolds for tissue engineering purposes has been shown
to offer significant clinical benefits [
]. The cell-material interaction is very complex and
seeded cells respond differently to specific surface chemistry and various architectural
parameters . In particular, the choice of scaffold material determines cell adhesion, which has
direct effects on cell behaviour including morphology and proliferation. Unchanged cellular
phenotype is an important aspect of biocompatibility. The cells show scaffold preferences and,
thus, the suitability of the material must be validated beforehand [42, 43]. The results of our
study indicate that Hs27 cells grow well on both investigated surgical scaffolds and show
apparently unchanged and typical fibroblast morphology. As for the proliferation rate, our
results demonstrate that attached Hs27 cells proliferate healthily on both investigated
materials. This is indicated by microscopical evaluation as well as by quantification of the metabolic
rate of the cells. To measure metabolism, we used a WST-1 test. Similar to the MTT method,
the WST-1 system is based on the conversion of tetrazolium to formazan. Another frequently
used system to study cell metabolism is based on the risazurin/resorufin conversion (e.g.
Alamar blue, CellTiterblue). However, as these systems measure fluorescence instead of
absorbance they tend to be influenced by fluorescent parts of the studied system and therefore we
preferred WST-1 in this case [
]. In our study, although we started with the same amount of
cells on all scaffolds, there were eventually more cells on Ethisorb scaffolds than on Vicryl. In
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Fig 6. Comparison of VEGF165 protein secretion from Hs27 cells grown on Vicryl and Ethisorb scaffolds. Human fibroblasts were
transfected with 20 μg VEGF165 plasmid DNA and co-cultured with Vicryl or Ethisorb scaffolds. After 1, 3, 6 and 9 days (d) the supernatant was
collected and the level of VEGF165 protein was determined by the enzyme-linked immunosorbent assay. Data shown are mean ± SEM from three
experiments with three technical replicates (n = 3; two-way ANOVA comparing VEGF165 concentration of cells grown on Vicryl and Ethisorb
scaffolds for each time point: **p<0.005, ****p<0.00005).
contrast to Vicryl, which has a woven scaffold-structure with small pores, Ethisorb consists of
nonwoven fibres resembling a fleece. This implicates that Ethisorb provides a higher surface
area compared to Vicryl allowing a greater number of cells to colonize and expand and, thus,
likely explains their greater metabolic activity on Ethisorb scaffolds. Another interesting
phenomenon we observed was that cells spread on the entire surface of the scaffolds and at the
later time points start to grow between the filaments of the respective materials. Due to the
manufacturing process of the materials there are relatively large pores (ca. 300μm) between the
respective cords of the Vicryl mesh, while the surface of Ethisorb mesh is dense. It seems that
this leads to a higher area for cell growth on Ethisorb, which may also explain the higher
VEGF expression with the Ethisorb mesh. Considering the restricted availability of autologous
fibroblasts for clinical use, the pore size of engineered scaffolds could therefore pose one of the
limiting factors for gene therapy-based soft tissue reconstructions.
Apart from the influence of the scaffold material on growth, proliferation and
differentiation of seeded cells, mechanical and degradation properties of biodegradable scaffolds may be
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affected by the cells [16, 45±48]. Surgical scaffold materials and tissue engineering constructs
must, first of all, provide sufficient mechanical support at the defect site. Ideally, the
mechanical properties of the scaffold material should not be affected by the cells which are, in a
preimplantation phase, grown on their surface. Therefore, in this study, we tested whether Hs27
cells influence the mechanical behaviour of Vicryl and Ethisorb scaffolds in the course of the
experiment. Of note, under our experimental conditions, fibroblasts were cultured as a
monolayer without adding extracellular matrix components. The results presented here show that
attached cells did neither lead to additional deterioration of the used scaffolds after 9 days nor
to protection of the fibers from hydrolytic degradation. Interestingly, Ethisorb lost mechanical
resistance much faster than Vicryl, although its components (much smaller pore size) should
theoretically degrade slower than the components of Vicryl [
The ability of cell therapy using genetically modified cells to deliver a bioactive protein at
the target site circumvents the limitations associated with conventional protein delivery
strategies. In this study, we made use of a well-established transfection method to generate
VEGF165-overexpressing fibroblast, which colonized efficiently the surface of Vicryl and Ethisorb
scaffolds. VEGF is a well-established therapeutic protein for enhancing angiogenesis both in
vitro and in vivo. Our and many other groups have previously shown that secreted VEGF can
enhance cell proliferation, wound healing and angiogenesis in different pre-clinical models
24, 49, 50
]. In present study, our main focus was not on the effect of VEGF on proliferation of
cells on scaffolds but rather if scaffolds can be used as carriers for cells which are genetically
engineered to secrete VEGF or other therapeutic proteins. The VEGF165 protein expression
profiles were similar between the investigated scaffolds showing a gradual decrease in protein
levels. The Hs27 cells grown on Ethisorb expressed higher levels of VEGF165 protein, probably
due to greater surface-to-volume ratio of Ethisorb scaffolds and higher number of attached
cells. In this study, Hs27 were transiently transfected with a VEGF165 expression plasmid.
This short-termed expression of VEGF165 protein is thought to stimulate biomaterial
vascularisation without negative consequences of prolonged angiogenic stimulation. Our results
show that the level of VEGF165 expression of Hs27 cells grown on scaffolds reaches the
therapeutic levels as demonstrated in the ischemic hind limb animal model [
]. There are concerns
about a possible oncogenic potential of genetically modified cells. These are mainly based
on applications with stable viral transfection, because this can induce oncogenic mutations
through random integration [
]. To avoid this problem, we have used here a plasmid vector
approach, which has an excellent safety profile, because the plasmid is not expected to be
integrated into the host genome and therefore the risk is much lower.
Although these in vitro findings present an important step toward construction of bioactive
PLGA-based gene delivery cell carriers, further investigations with different therapeutic
proteins are needed to determine their clinical utility. Moreover, although it is important to test the
in vitro methods in vivo, we think it is not necessary to perform the in vivo study for the current
in vitro method. First, several former studies including ours have already proven the use of
VEGF as a therapeutic protein for improving angiogenesis and wound healing both in vitro and
in vivo [
24, 48, 49
]. Moreover, we have published a pre-clinical study regarding the
geneticmodified fibroblasts expression angiogenic factors including VEGF. In this work we actually
applied the same transfection method with fibroblasts and VEGF plasmid as in the current one.
We found that genetically modified fibroblasts can enhance angiogenesis and arteriogenesis in
a hindlimb ischemia model [
]. This study was a proof-of-concept for our transfection method
and in vivo application. Also, both PLGA-based meshes used in our study are clinical level
products used for decades. Thus, our current study is mainly focused on the evaluation of a possible
carrier material for such applications. We show here that the cells not only are able to survive
and proliferate on the scaffolds but more importantly also produce the desired protein.
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In summary, in the present study, we show that human fibroblasts seeded on biodegradable
Vicryl and Ethisorb scaffolds show excellent biocompatibility. Furthermore, this model system
allows successful genetic modification of the cells. The presented methodology could be easily
adapted for other proteins and growth factors allowing a broader use of this gene-enhanced
Bioresorbable PLGA scaffolds can be used as vehicle for the delivery of transiently transfected
cells and may open the way for a variety of applications of gene therapy, tissue engineering and
regenerative medicine. Scaffolds with a condensed structure and smaller pore size might lead
to a better cell-scaffold interaction and thus lead to a higher yield of the desired recombinant
S1 Data. Raw data of mechanical property test.
S2 Data. Raw data of cell proliferation test with WST-1.
A big thank you to Eduardo Grande for supporting the mechanical testing. We also thank
Simone Schmalix for her excellent technical assistance. Dr. Ziyang Zhang would like to
personally thank Dr.Lumimomo for her mental support during the preparation of the manuscript.
Conceptualization: AFS TP ZZ RHB MG.
Data curation: TP ZZ PF.
Formal analysis: TP UH ZZ PF.
Funding acquisition: AFS ZZ HGM MG.
Investigation: TP ZZ UH PF AS.
Methodology: TP ZZ UH KK AS.
Project administration: AFS.
Resources: AFS MG.
Software: ZZ TP.
Visualization: ZZ TP UH KK.
Writing ± original draft: ZZ TP.
Writing ± review & editing: AFS ZZ.
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