electrospun biomimetic microfibers for pelvic floor repair ...€¦ · options for recurrence of...
TRANSCRIPT
Electrospun biomimetic microfibers for pelvic
floor repair tested in a rat abdominal wall
defect model
Cecilie Lærke Glindtvad [email protected]
Tel: +45 20978986
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Resumé
Introduktion: Fra 50 års alderen vil halvdelen af den kvindelig befolkning opleve nedsynkning i
underlivet pga. udtynding og bristning i støttende bindevæv og fascier i bækkenet. Der er ingen
fuldstændig effektiv behandling for nedsynkning i underlivet, som giver et varigt resultat og er uden
komplikationer. Vi forslår en ny behandlingsmetode med rekonstruktion af de tre dimensionale
anatomiske strukturer i bækkenbunden.
Formål: At undersøge en nedbrydelig mesh med fibroblast vækstfaktor (bFGF) i forhold til mekaniske
egenskaber og effekt på collagen og elastin dannelse i nyt bindevæv.
Materiale og metode: 40 rotter fik indopereret en elektrospundet polycaprolactone(PCL)/
polyethylene oxid (PEO) mesh af mikrofibre. Test gruppen havde bFGF i kernen af mesh fibrene, og
hos kontrol gruppen var bFGF ikke tilsat den indopererede mesh. Meshen blev testet i en abdominal
defekt model i rotterne. Testområdet blev udtaget efter 4, 8 og 24 uger og testet for styrke,
forlængelse, stivhed, energi absorption, total collagen mængde, elastin samt mRNA expression og
protein mængde af collagen-I, collagen-III og fibronectin. Den samlede effekt af meshen blev vurderet
ud fra størrelsen af det hernie, som en stor del af rotterne udviklede.
Resultater: Studiet viste en forøgelse af styrken, stivheden og energien over tid. Desuden sås en
signifikant forøgelse efter 4 uger i gruppen med bFGF for styrke, stivhed og energi absorption. Der var
ingen forskel mellem grupperne efter 8 og 24 uger. Der var en signifikant forøgelse af mRNA
expression for collagen-I og collagen-III efter 4 uger. Der blev for grupper med og uden bFGF ikke
fundet en forskel for protein mængden, elastin eller total collagen. Histologien viste en hæmmet celle
indvækst ved 4 uger med bFGF, men ved 8 og 24 uger var der ingen forskel. Desuden sås tegn på
elastin fibre ved 24 uger. Analyse af hernie størrelse i forhold til variablerne viste, at øget hernie
størrelse medførte øget styrke og stivhed.
Konklusion: Analyserne viste at en mesh af PCL/PEO fremmer dannelse af nyt væv med øget styrke,
stivhed og energi absorption over tid. Meshen med bFGF havde kun en effekt efter 4 uger på styrke,
stivhed, energi absorption og mRNA expression for collagen-I og collagen-III. Der var øget styrke og
stivhed med stigende hernie størrelse.
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Introduction
Half of the female population experiences disorders of the pelvic floor caused by weakening and
rupture of supportive connective tissue sheet or fascia [1]. This disorder can lead to pelvic organ
prolapse (POP) seen as a herniation of the anatomical structures (bladder, uterus and intestines) into
the vagina [2]. The frequency of POP generally increases with age and affects quality of life in terms of
social, psychological, physical, sexual, body image, and overall wellbeing [3]. Consequently, these
problems occupy a significant proportion of the healthcare budget now and expenses will continue in
the future [4].
POP affects up to 50% of all women over the age of 50 [5]. The mean prevalence is 19.7%, ranging
from 3.4-56.4% in developed countries [6]. The lifetime risk for undergoing POP surgery is estimated to
11.1-19% [5,7]. Furthermore, the risk of reoperation because of recurrence is up to 30% [8].
Recently, no efficient treatment without side effects or risk of recurrence has been invented. Instead,
each country and sometimes each hospital in a country use different operations and treatment-
techniques. So far, surgeons all over the world have tried to treat POP operatively with native tissue
repair, biological graft, and synthetic mesh in different shapes and sizes.
Synthetic, non-degradable mesh made of polypropylene (PP), has been used for operative correction
of complicated POP cases and recurrence occurs in up to 9.7% of cases [9-11]. Moreover, serious
complications of non-degradable meshes are seen with poor tissue integration, inflammation,
exposition of the mesh through the vaginal wall, and erosion into the bladder or bowel [12].
Consequently, usage of non-degradable mesh is reduced, even though there are few other treatment
options for recurrence of POP. Therefore, new principles for effective repair or replacement of
deficient connective tissue are needed. Collagen I, III, and V are the principal components of the
connective tissue in the pelvic floor providing strength to the soft tissue [13]. Furthermore, the pelvic
organs rely on the tensile strength of the pelvic floor for support; in this matter elastin is a part of
extension, resilience and recoil of tissue.
In recent years, approaches based on tissue engineering and regenerative medicine principles have
emerged as strategies for pelvic tissue reconstruction [14,15]. In this matter, we suggest that
treatment of pelvic floor disorders could be brought forward by functional reconstruction of the three
dimensional, anatomical structures by deploying a degradable, electrospun mesh. Our approach
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includes release of basic fibroblast growth factor (bFGF) with the purpose to enhance the formation of
new connective tissue, and in particular collagen deposition within the degrading mesh [16]. An
engineering challenge is to balance mesh degradation and implant weakening with the increase of
structural stability accompanying collagen deposition. In vitro studies show that a polycaprolactone
(PCL) /poly ethylene oxide (PEO) mesh with bFGF increases fibroblast cell proliferation with 331% after
9 days of culture, and that 72% of the bFGF is released. Studies show that the PCL/PEO fibers without
bFGF have no toxic effect and that added bFGF increases cell viability [17]. The results of the former in
vitro study indicated that a PCL mesh with bFGF could be a potential treatment for pelvic organ
prolapse and was chosen to test in vivo.
The purpose of this study is to investigate the in vivo effects of a degradable, bFGF releasing,
electrospun mesh on collagen and elastin production and study the influence on the temporal
mechanical properties of the neotissue in a rat abdominal wall defect model.
Materials and Methods
The mesh was made of biodegradable, biocompatible PCL/PEO microfibers (97% PCL and 3% PEO) and
consisted of randomly placed hollow fibers with bFGF in the core. The microfibers were continuous
with high porosity and high spatial interconnectivity, which mimicked the intricate interweaving
structure of extracellular matrix composed of fibrillar collagen and elastin promoting cell adhesion. The
microfibers were produced by coaxial electrospinning, fabricating two-component fibers by forcing
solutions through a coaxial spinneret in the presence of a high electric field. The fibers consisted of PCL
in the outer wall, polyethylene oxide (PEO) and bovine serum albumin (BSA) in the inner wall, and
bFGF in the core. The fibers in the present mesh measured 1.6 µm in shell thickness and had a fiber
diameter of 7.489 ± 0.445 µm. The amount of bFGF in the mesh was 60 ng bFGF per mg mesh
corresponding to approximately 0.34851 µg bFGF per implanted mesh.
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Figure 1 – Scanning electronic picture of the mesh. Cross-section (a) and surface (b) for mesh without bFGF. Cross-section (c) and surface (d) for mesh with bFGF [17].
Mesh with bFGF was not visibly morphologically different from mesh without bFGF as shown in Figure
1. Three-dimensional structure of the mesh and thereby its ability to mimic the extracellular matrix
composition was not changed with bFGF in the core [17].
Implantation
Forty inbred Wistar female rats (adult, weight 250–300 grams) were purchased from Taconic,
Denmark. A pilot study included eight rats and the primary study included 32 rats. Half of the rats
served as a control group with mesh without bFGF and the other half as test group with bFGF. After an
abdominal midline incision, the skin was loosened from the muscle layer and a full thickness fascia-
muscle defect (1.5 cm x 3.0 cm) was made. Then, the defect was repaired by the mesh with a 0.5 cm
overlap to the surrounding tissue in all directions. The mesh was independently fixated with single
sutures (Prolene 5-0, Ethicon®) in the corners and with continuously absorbable sutures (Vicryl 4-0
Ethicon®) at the peripheral edges (Figure 2). The skin was closed by staples.
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Figure 2 – Overview of the mesh and explantation schedule. The grey square is the mesh, the red square is the muscle defect, the dashed squares is the pieces for mechanical testing, and the blue lines are the permanent stitches.
From implantation to explantation of the mesh the operation area was observed for herniation, fluid
collection and complications in general. During the study, one rat died because of peritonitis (24 weeks
+ bFGF) and two rats were sacrificed: one because of infection in the operation wound (24 weeks –
bFGF) and one because of suspicion of incarcerated hernia (8 weeks + bFGF).
Explantation
After four, eight, and 24 weeks rats were sacrificed by Pentobarbital overdose (1 ml of Pentobarbital®
200 mg/ml with 10% Lidocaine® intraperitoneally) (Table 1).
4 weeks 8 weeks 24 weeks In total
Mesh without bFGF 4 rats 6 rats 9 rats 19 rats
Mesh with bFGF 4 rats 5 rats 9 rats 18 rats
Table 1 - Time schedule for explantation
The implantation area with one cm surrounding tissue was excised and divided into four sections. One
section for total collagen, elastin, and mechanical testing which was wrapped air-tight and stored at
minus 20 degrees. Two sections were snap frozen in liquid nitrogen and used for evaluating mRNA
expression and protein content. The fourth section was formalin fixed and used for histology (Figure
2).
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Mechanical testing
After thawing, the material was kept moistened with a 50 mM Tris/HCl (pH 7.4) buffer. Four mm wide
strips were cut with a multicutter (parallel mounted razor blades) and adjusted to 18 mm in length.
The thickness of the central part of the strip was measured by an electronic length gauge (MT25/ND
281B, Heidenhain, Traunreut, Germany) under standardized stress (25 kPa) for 10 seconds. For
mechanical testing, the ends of the strip were gripped in clamps mounted with 10 mm gage distance in
a material testing machine (Alwetron TCT5, Kista, Sweden) equipped with a 20 N load cell. To avoid
slippage the clamp jaws were coated with emery cloth (grain size 320) and screwed together to an
adjusted and standardized moment with a torque wrench (22 cNm). During testing at 30 mm/min the
strip was soaked in the Tris/HCL buffer. The strip was subjected to loading until failure. The ruptured
pieces were collected for total collagen analysis.
mRNA expression by real-time PCR
mRNA was extracted from tissue flaps using Trizol reagent (Invitrogen). For each sample 0.5 µg RNA
was reverse-transcribed using RT-Superscript III (Invitrogen) and polymerase chain reaction (PCR) was
carried out by using iCycler™. All the reactions were performed with SYBR® Green under the same
conditions as follows: 95°C for five minutes followed by 45 cycles 30 seconds at 95°C , one minute at
60°C , 45 seconds at 72°C , and five minutes at 72°C. A standard curve was mixed from all the copy
DNA samples and included in each PCR dilution (1:5, 1:25, 1:125, 1:625). To quantify the expression of
the genes, they were compared to the housekeeping gene Glyceraldehyde 3-phosphate
Dehydrogenase (GAPDH), and the results were expressed as a ratio. The primer pairs used in the study
were: forward collagen-I 5’-3’ ATG TTC AGC TTT GTG GAC CT, reverse collagen-I 5’-3’ CAG CTG ACT
TCA GGG ATG T, forward collagen-III 5’-3’ CGG AAT TGC AGA GAC CTG AA, reverse collagen-III 5’-3’
ACA GTC ATG GGA CTG GCA TTT AT ,forward fibronectin 5’-3’ GTG GCT GCC TTC AAC TTC TC and
reverse fibronectin 5’-3’GTG GGT TGC AAA CCT TCA AT.
Protein content by Western-blots
Total protein was extracted from the tissue samples by homogenization using a homogenization buffer
containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), protease and phosphatase
inhibitors. Each sample was homogenized with 20 µl buffer/mg wet tissue and ceramics beads on a
Precellys. The protein concentration was determined by the Micro BCA Protein Assay kit (Pierce).
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Running samples were mixed in Laemmli Buffer and 20µg sample was loaded on a 4-15% gel Criterion
TGX Stain-Free BioRad. Rainbow marker (2.5µl) was used. The gel was run on a vertical electrophorese
(SDS-Page). After 2.5min UV activation of the gel the protein was transferred to a membrane using
BioRad TurboTrans HIGH MW. After transfer, a stainfree picture of the membrane was made and used
for detection of reference protein as total protein on the membrane instead of using beta-actin
concentration as a reference [18]. After blocking of the membrane it was blotted with primary
antibodies for Collagen-I (Abcam, cat.no: ab90395, 1:1000), Collagen-III (GenWay Biotech cat.no:
292343, 1:2500) or Fibronectin (DAKO cat.no: A0245, 1:5000) over night. Afterwards, the membranes
were washed inTTBS buffer before placing in secondary antibody; Collagen-I (anti-mouse SC-2005,
1:1000), Collagen-III (anti-rabbit SC-2054, 1:5000) and fibronectin (anti-rabbit SC-2054, 1:10.000).
Again, the membranes were washed before the induction with SuperSignal West Dura Extended
Duration Substrate (Pierce) on Imagelab (BioRad). The procedure for collagen-I was not successful and
ELISA was used instead.
ELISA
100µl of serial dilutions of Collagen l standard (from 12.5 to 0.39 ng/ml) diluted in PBS, (cat:1200-01
Southern Biotechnology, USA) and 100µl of serial dilution of sample (1:4000 to 1:160000 ) diluted in
PBS was coated in an ELISA microplate and incubated overnight at RT. After blocking for 30 minutes
with 5% Milk in PBST the plate was washed and incubated for 90 minutes at RT with 100µl 1:1000
dilutions of goat-anti-collagen l biot. (cat: 1310-08, Southern Biotechnology, USA) Wells were washed
and incubated with 100µl 1:1000 Streptavidin-HRP (cat:7100-05, Southern Biotechnology, USA). The
wells were washed before the induction with TMB substrate (Cat: TMBW-100, SMS, Denmark). The
reaction was stopped with 0.2M H2SO4. The Optical Density was determined at 450nm microplate
reader. Standard absorbance versus its concentration was plotted on graph to construct a standard
curve. The result is shown as a ratio between Collagen l and total protein. Results for four weeks
samples were not achieved because of lack of test material.
Total collagen content
The collected strips from mechanical testing were defatted in acetone 2 × 1 hour at room temperature,
air-dried and weighed. To remove the remaining PCL in the strips, they were treated with chloroform:
methanol 2:1, for 21 hours at 4°C, air-dried, and weighed. Before hydrolysis in 6 N HCl at 100°C for 16
hours, the strips were freeze-dried and weighed. Hydroxyproline (Hyp) was determined colorimetric
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[19]. Collagen content was calculated as Hyp × 7.46 [20]. The total collagen amount was correlated to
the cross-sectional area of the samples [mg/mm2] to neutralize differences in sample size.
Histology
The samples for histology were fixated in four percent neutral formaldehyde buffer for 24-48 hours.
The formalin fixed paraffin embedded tissue was cut in 3 µm thick slices, collected on slides, treated in
tissueclear and stored in water until staining. Sections were stained with Hematoxylin Eosin performed
on a Combitec slide stainer 4009 from HISTOLAB Products, Göteborg Sweden and Masson’s trichrome
staining was performed with the three colors; Harris Hemotoxylin, Ponceau Red and Anilin Bleu. After
staining, the sections were dehydrated and fixated with the mounting medium Pertex. After staining,
the samples were blinded within the groups of four, eight, and 24 weeks and investigated for visual
differences in relation to amount of mesh left, collagen amount and inflammatory response.
Ethical
The study was approved by the Danish Inspectorate for Animal Experimentation under the Danish
Ministry of Justice with authorization number 2014-15-2934-01021. The animals were anesthetized
and treated with analgesics according to ethical guidelines and looked for by professional animal
keepers under guidance from a veterinarian.
Statistics
The number of animals was chosen from former short term studies with an identical animal model
[21,22]. Data were tested for normality. Differences between groups were assessed by Student’s t-test.
The mechanical testing was expressed in mean results calculated from one to five test samples for
each rat. The cross-sectional area and volume used, was the one priori to testing. Furthermore, the
removed abdominal wall from the implantation of the mesh was used as a reference and a goal for the
mechanical properties for the newly formed tissue. The mesh values were reference for the starting
point for the mechanical properties. Data were analyzed for association between hernia size and the
various outcomes by two-way ANOVA. Spearmen’s Rho was performed when a trend seemed possible.
The statistic program StataIc version 13 was used. Significance level: P ≤ 0.05.
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Results
The mechanical tensile tests are presented as a mean stress-strain curve for each group (Figure 3).
Strain is fractional increase ((l-l0)/l0) in sample length (l) where l0 is the length of the sample at a
recorded minimal load value (0.003 N). The vertex of the curve is the maximum stress representing the
tensile strength of the sample, given as the force measured by the load cell divided by the initial cross
sectional area of the sample. The maximum slope of the stress-strain curve represents the maximum
stiffness of the specimen. The area below the stress-strain curve until failure represents the energy
absorption needed for the tissue deformation.
Figure 3 – Stress-strain curve of mean values for each group, mesh, and explanted muscle. The work curve for the mesh continues to break at mean strain 8.70 (870%) and stress at 2.1 MPa. Four animals in the two four weeks groups, Five and six rats in the eight weeks groups and nine rats in each of the 24 weeks groups.
Figure 4 shows tensile strength (P=0.0021), stiffness (P=0.0002), and energy absorption(P=0.0097) in
the newly formed tissue with a significant difference between four weeks samples with and without
bFGF, but no difference at eight and 24 weeks. For strain at tensile strength, no differences at any
point between the groups with and without bFGF were seen. For all parameters except strain at tensile
strength, there was a significant difference over time from four to 24 weeks (P<0.0002) (Figure 4).
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Figure 4– Bar graph of tensile strength, stiffness, energy absorption, and strain at tensile strength in the newly formed tissue.
A significant increase in mRNA expression for collagen-I (P= 0.0060) and collagen-III (P= 0.0086) was
seen in the group with bFGF at four weeks. This difference was equalized at eight and 24 weeks (Figure
5). For fibronectin mRNA expression there was only a significant increase at 24 weeks in the group
without bFGF (P= 0.0104) (Figure 5).
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Figure 5–Bar graphs for collagen-I, collagen-III, and fibronectin mRNA expression, comparing groups with and without bFGF at four, eight and 24 weeks.
No difference between groups with and without bFGF for protein amount for collagen-I, collagen-III
and fibronectin was seen at any time.
For total collagen amount in relation to cross-sectional area no significant difference between the
mesh with and without bFGF was seen. But over time, there was a significant increase in the collagen
amount (P=0.0000) (Figure 6).
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Figure 6–Total collagen amount in relation to
cross-sectional area.
Histologically, the Weigert’s elastin stain showed no elastin fibers in the newly formed tissue at four
and eight weeks. At 24 weeks there were areas of elastin in eight of 18 samples independent of bFGF
status but five of the eight samples were from rats with bFGF (Figure 7).
Maisson trichrome staining showed that the four weeks samples with bFGF had a clear blue area of
collagen at the edge of the sections (Figure 8A). The mesh was seen as an area of light pink with
collagen ingrowth at the edge (Figure 8a). In the four weeks samples without bFGF no clear area of
mesh was seen, instead small stripes of mesh surrounded by cells were seen. Figure 8B shows
ingrowth of collagen throughout almost the entire depth of the mesh. Equally, all the four weeks
samples presented giant cells with multiple nuclei (Figure 8b). At eight weeks, there was no clear
difference between the sections with and without bFGF (Figure 8 C and D), but clear fibers of collagen
and multiple giant cells (Figure 8 c and d). After 24 weeks still no difference between with and without
Figure 7 – Representative cross section of 24 samples with and without bFGF. Weigert’s elastin stain - Magnification x40 with scale bar indicating 100 µm. Blue-black fibers in the left of the picture are elastin fibers.
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bFGF was seen (Figure 8 E and F), instead there was a decrease in the number of giant cells and an
increase in the amount of collagen fibers compared to eight weeks samples (Figure 8 e and f).
Figure 8 – Representative cross section of four (A and B), eight (C and D) and 24 weeks (E and F) samples with and without bFGF. Masson’s Trichrome staining; A, B, C, D, E, and F are magnification x10 with scale bar indicating 200 µm. a, b, c, d, e, and f are magnification x40 with scale bar indicating 100 µm.
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The results of 24 weeks samples were categorized according to hernia size no matter if they were with
or without bFGF (Table 2). There was a significant increase of tensile strength and stiffness with
increasing hernia size, and for fibronectin mRNA expression a decrease with increasing hernia size was
seen (Figure 9). For the remaning variables no difference was oberserved (Table 3).
Category Hernia size
Group 1 None Group 2 Up to 3x3 cm Group 3 Over 3x3 cm
Table 2 – Categorization of hernia size.
P-value
Tensile strength [MPa] 0.0118 Strain at tensile strength 0.0867 Stiffness [MPa] 0.0214
Energy absorption [mJ/mm3] 0.1730
Total collagen amount [mg/mm2] 0.4078 mRNA – Collagen-I 0.2667 mRNA – Collagen-III 0.0902 mRNA - Fibronectin 0.0126 Protein – Collagen-I 0.6722 Protein – Collagen-III 0.1548
Table 3 – P-values for correlation between tensile strength, strain, stiffness, energy absorption, mRNA expression, protein amount, and total collagen amount in relation to hernia size.
Figure 9– Tensile strength, stiffness, and fibronectin mRNA expression in relation to hernia size. Continued. .
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Figure 9 continued – Tensile strength, stiffness, and fibronectin mRNA expression in relation to hernia size.
Discussion
The present study investigated a degradable PCL/PEO mesh from a mechanical point of view but also
regarding collagen and elastin formation in the newly formed tissue. The idea was to test a new mesh
treatment for POP in an animal model with the future aspect to be able to treat POP in women. The
optimal outcome of an in vivo mesh study would be strong, elastic neotissue with growth of collagen
suitable for treatment of POP. By adding bFGF to the mesh, an in vitro study showed enhanced
fibroblast proliferation with a promising rate of formation and sufficient supportive mechanical
properties of the new connective tissue [17]. The effect of the mesh in the present study was explored
by mechanical testing, histology, total collagen amount and analysis of mRNA expression and protein
amount of collagen-I, collagen-III and fibronectin. These components were considered the most
important in strong connective tissue. Despite the hypothesis based on promising in vitro studies, we
did not succeed to show significant effect of adding bFGF to the mesh in our in vivo study. But the
mesh itself showed an effect on tissue formation.
Experimental strengths and weaknesses
This study is a rethinking of POP treatment with a three dimensional repair of the pelvic floor with
formation of new connective tissue. Hereby, long-term complications from foreign bodies as seen with
non-degradable meshes were reduced. Another strength was the long follow-up time with results at
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eight and 24 weeks preceded by a four weeks pilot study. The pilot study showed no indications for
changing the design of the animal model. Without the long follow-up time we might not have seen the
effect of bFGF wear off.
The animal model itself had both strength and weakness. The mesh was tested as a patch over a full
wall muscle defect on the abdomen of the rat which gave the opportunity to explant only newly
formed tissue for analysis. The weakness was that the mesh was implanted on the abdomen of a
rodent walking on four legs. In the “real world” a mesh would be used for women walking in an upright
position with a heavy load on mesh and pelvic floor. Furthermore, a mesh on the abdomen was
surrounded by muscle instead of connective tissue, which can influence the results negatively.
The small number of animals in each study group was a weakness, that could have affected and
underestimated the results. A larger number of rats in each group might have shown more significant
results than we did. Nevertheless, the total number of rats was large compared to similar studies
[21,22]. In relation to the use of bFGF, the release of approximately 75% of the bFGF in 9 days as seen
in in vitro studies could be a problem not knowing when the last 25% was released in our in vivo study
[17]. A steady state release over a longer period of time would be preferable. However, the release
profile in vitro was hard to mimic in the in vivo environment.
The usage of bFGF
Despite in vitro studies where bFGF enhanced fibroblast cell proliferation, our in vivo study did not
show the same effect, at least judged from accumulation of cells. The only effect of bFGF was seen
after four weeks in collagen-I and collagen-III mRNA expression, tensile strength, strain, stiffness, and
energy absorption.
The detectable effect on mRNA level but not on protein level could be explained by the short time of
bFGF release with a peak release of 10.6% of the bFGF in the first hour and 52.9% during the following
day one to six [17]. In addition, there was a histologically difference between the two four weeks
groups. It seemed as if bFGF inhibited cell accumulation and collagen deposition within the mesh. This
effect was difficult to explain because in vitro results suggested that the mesh was non toxic and that
bFGF increased fibroblast cell proliferation [17].
Heybeli et al used an identical animal model and tested the effect of bFGF loaded polyprolene mesh
compared to polyprolene mesh without bFGF. After four weeks they showed no difference between
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the groups. After eight weeks there was an increase in tensile strength in the group with bFGF but also
increased vascularization, a higher amount of fibroblasts, and a greater amount of collagen fibers in
the bFGF group than the group without bFGF [22].
The difference between the study by Heibeli et al. and the present study could be due to different
dosages of bFGF and a difference in the release profile. We used 0.34851 µg bFGF per implanted mesh
and Heybeli et al. used 4-12 times more in the dosage of bFGF. [22] Furthermore, 75% of the bFGF
dosage used in this study was released in 9 days in vitro [17]. From our in vitro study, we know that
our mesh had a burst release of bFGF, and the release profile in the study by Heybeli et al. is not
described.
In our study, the histology showed a clear difference at four weeks, where bFGF seemed to inhibit cell
popularization and collagen deposition within the mesh. The inhabitation was reflected on the
mechanical results where the four weeks samples with bFGF resembled the results of the mesh.
Thereby, the four weeks samples with bFGF were significant stronger and stiffer than the samples
without bFGF. The higher stiffness results from less scaffold degradation in this case and thus, the
presence of bFGF seamed to alter not only collagen deposition but also scaffold degradation by giant
cells.
Roman et al. investigated the use of adipose-derived stem cells (ADSC) and oral fibroblasts on poly-L-
lactic acid (PLA) scaffolds for tissue engineered POP repair material.[23]. They found that ADSC
resulted in more total collagen than oral fibroblasts. Under restrained conditions, the two cell types
enhanced the same collagen amount and an increased elastin production indicating that both cell
sources were proper candidates for tissue engineering. We tested mesh with bFGF. Comparing our
minimal effect on collagen formation and the promising results of Roman et al., stem cells in general
seem to be a better choice for the future research in the field.
Rate of new tissue formation and mesh degradation
The mesh alone was the stiffest material of the examined samples, followed by the four weeks
samples with bFGF, 24 weeks samples, muscle from the implantation site, and last 8 and 4 weeks
samples without bFGF. This might reflect that the mesh was too stiff compared to the muscle. The
mesh was also stronger than the muscle and any of the test groups. From comparing work curves in
figure 3 it was seen that in the low strain domain ( < 0.1 strain) a toe region existed in the degraded
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meshes and muscle tissue, which was not present in the initial mesh. The consequence was, that cells
situated within muscle tissue or degraded meshes were thus subjected to higher strains at the same
load, compared to cells in the initial mesh. It is known that mechano transduction influences cell
maturation and growth [24], and that dynamic stretching alter adhesion, proliferation, locomotion,
morphology, and the synthetic profile of cells [25]. Additionally, the work curve of the mesh did not
show a vertex at approximately 0.5 strain as all other samples did. The later was not expected to
influence cell dynamics as in vivo loads never implied such high strain levels. But these factors could
indicate that the mesh had the wrong mechanical properties in comparison with the target tissue and
therefore led to herniation.
Finally, the work curve shows that the newly formed tissue was the most fragile at eight weeks, in
consistence with the start of herniation at nine weeks. This could indicate that the degradation of the
mesh was too fast in comparison with the formation of the new connective tissue leading to a period
where herniation could appear. At 24 weeks, the strength of the tissue increased to the level of the
four weeks samples without bFGF indicating that a formation of new tissue could have strengthened
the area of the mesh.
Herniation
In the animal study, it was possible to investigate the factors important for developing or preventing
hernia when dealing with newly formed connective tissue. The categorization of the degree of
herniation into three groups made it possible to test the impact of the measured variables on hernia
formation. The results showed that only tensile strength and stiffness were increased with increasing
hernia size. Former studies investigating samples from the pelvic floor from women with and without
POP showed that women with POP have weaker and stiffer tissue than women without POP [26,27].
These results were supported by the findings of the present study were we expected that dynamic
loading at low strain levels would lead to increased collagen deposition. Meaning, development of a
mesh which enhances formation of new strong and elastic tissue seems to be a theoretical possible
treatment of POP. The design of a toe-region in the stress-strain curve of the mesh together with an
improved and sustained release of growth factor is a strategy to be tested.
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Conclusion
A tissue-engineered treatment for POP with a degradable PCL/PEO mesh could be possible judged
from the results of the present animal model. The degradable mesh promoted tissue formation with
increasing collagen amount and biomechanical mesh properties being temporal adjusted toward the
behavior of muscle. However, the addition of bFGF (60 ng bFGF per mg mesh) did not add any long-
term positive effects. The occurrence of hernias accompanied with an increase in total collagen,
strength and stiffness over time, leads to an assumption of a mismatch between the degradation time
of the mesh and the formation of the new tissue. The results suggest that the degradation of the mesh
is too fast compared to the formation of new tissue and thereby leading to herniation. Regarding the
hernia analysis, the mesh should not be too stiff since such an environment seems to limit collagen
formation and consequently increase the strength and stiffness of the new tissue. The outcome can be
improved through mesh redesign incorporating tailored mechanical properties and bFGF release to
match mesh degradation time with a tissue formation that will prevent herniation.
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